Compositions and methods for treating CEP290-associated disease

ABSTRACT

Compositions and methods for treatment of CEP290 related diseases are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of U.S. patentapplication Ser. No. 15/667,603, filed Aug. 2, 2017, which is acontinuation-in-part of U.S. patent application Ser. No. 14/644,181,filed Mar. 10, 2015, now U.S. Pat. No. 9,938,521, issued Apr. 10, 2018,and claims the benefit of U.S. Provisional Appl. No. 61/950,733, filedMar. 10, 2014; U.S. Provisional Appl. No. 62/036,576, filed Aug. 12,2014; U.S. Provisional Appl. No. 62/370,202, filed Aug. 2, 2016; U.S.Provisional Appl. No. 62/400,526, filed Sep. 27, 2016; U.S. ProvisionalAppl. No. 62/443,568, filed Jan. 6, 2017; U.S. Provisional Appl. No.62/503,800, filed May 9, 2017; and U.S. Provisional Appl. No.62/535,193, filed Jul. 20, 2017; the contents of which are herebyincorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 2, 2018, isnamed SequenceListing.txt and is 1,535,149 bytes in size.

FIELD OF THE INVENTION

The invention relates to CRISPR/CAS-related methods and components forediting of a target nucleic acid sequence, and applications thereof inconnection with Leber's Congenital Amaurosis 10 (LCA10).

BACKGROUND

Leber's congenital amaurosis (LCA) is the most severe form of inheritedretinal dystrophy, with an onset of disease symptoms in the first yearsof life (Leber 1869) and an estimated prevalence of approximately 1 in50,000 worldwide (Koenekoop 2007; Stone 2007). Genetically, LCA is aheterogeneous disease. To date, fifteen genes have been identified withmutations that result in LCA (den Hollander 2008; Estrada-Cuzcano 2011).The CEP290 gene is the most frequently mutated LCA gene accounting forapproximately 15% of all cases (Stone 2007; den Hollander 2008; denHollander 2006; Perrault 2007). Severe mutations in CEP290 have alsobeen reported to cause systemic diseases that are characterized by braindefects, kidney malformations, polydactyly and/or obesity (Baal 2007;den Hollander 2008; Helou 2007; Valente 2006). Mutations of CEP290 areobserved in several diseases, including Senior-Loken syndrome, MeckelGruber syndrome, Bardet-Biedle syndrome, Joubert Syndrome, and LeberCongenital Amaurosis 10 (LCA10). Patients with LCA and early-onsetretinal dystrophy often carry hypomorphic CEP290 alleles (Stone 2007;den Hollander 2006; Perrault 2007; Coppieters 2010; Littink 2010).

LCA, and other retinal dystrophies such as Retinitis Pigmentosa (RP),have long been considered incurable diseases. However, the first phaseI/II clinical trials using gene augmentation therapy have led topromising results in a selected group of adult LCA/RP patients withmutations in the RPE65 gene (Bainbridge 2008; Cideciyan 2008; Hauswirth2008; Maguire 2008). Unilateral subretinal injections ofadeno-associated virus particles carrying constructs encoding thewild-type RPE65 cDNA were shown to be safe and moderately effective insome patients, without causing any adverse effects. In a follow-up studyincluding adults and children, visual improvements were more sustained,especially in the children all of whom gained ambulatory vision (Maguire2009). Although these studies demonstrated the potential to treat LCAusing gene augmentation therapy and increased the development oftherapeutic strategies for other genetic subtypes of retinal dystrophies(den Hollander 2010), it is hard to control the expression levels of thetherapeutic genes when using gene augmentation therapy.

LCA10, one type of LCA, is an inherited (autosomal recessive) retinaldegenerative disease characterized by severe loss of vision at birth.All subjects having LCA10 have had at least one c.2991+1655A to G(adenine to guanine) mutation in the CEP290 gene. Heterozygous nonsense,frameshift, and splice-site mutations have been identified on theremaining allele. A c.2991+1655A to G mutation in the CEP290 gene giverise to a cryptic splice donor cite in intron 26 which results in theinclusion of an aberrant exon of 128 bp in the mutant CEP290 mRNA, andinserts a premature stop codon (P.C998X). The sequence of the crypticexon contains part of an Alu repeat.

There are currently no approved therapeutics for LCA10. Despite advancesthat have been made using gene therapy, there remains a need fortherapeutics to treat retinal dystrophies, including LCA10.

SUMMARY OF THE INVENTION

The inventors have addressed a key unmet need in the field by providingnew and effective means of delivering genome editing systems to theaffected tissues of subjects suffering from CEP290 associated diseasesand other inherited retinal dystrophies. This disclosure providesnucleic acids and vectors for efficient transduction of genome editingsystems in retinal cells and cells in other tissues, as well as methodsof using these vectors to treat subjects. These nucleic acids, vectorsand methods represent an important step forward in the development oftreatments for CEP290 associated diseases.

In one aspect, the disclosure relates to a method for treating oraltering a cell in a subject (e.g., a human subject or an animalsubject), that includes administering to the subject a nucleic acidencoding a Cas9 and first and second guide RNAs (gRNAs) targeted to theCEP290 gene of the subject. In certain embodiments, the first and secondgRNAs are targeted to one or more target sequences that encompass or areproximal to a CEP290 target position. The first gRNA may include atargeting domain selected from SEQ ID NOs: 389-391 (corresponding RNAsequences in SEQ ID NOs: 530, 468, and 538, respectively), while thesecond gRNA may include a targeting domain selected from SEQ ID NOs:388, 392, and 394 (corresponding RNA sequences in SEQ ID NOs: 558, 460,568, respectively). The Cas9, which may be a modified Cas9 (e.g., a Cas9engineered to alter PAM specificity, improve fidelity, or to alter orimprove another structural or functional aspect of the Cas9), mayinclude one or more of a nuclear localization signal (NLS) and/or apolyadenylation signal. Certain embodiments are characterized by Cas9sthat include both a C-terminal and an N-terminal NLS. The Cas9 isencoded, in certain embodiments, by SEQ ID NO: 39, and its expression isoptionally driven by one of a CMV, EFS, or hGRK1 promoter, as set out inSEQ ID NOs: 401-403 respectively. The nucleic acid also includes, invarious cases, first and second inverted terminal repeat sequences(ITRs).

Continuing with this aspect of the disclosure, a nucleic acid comprisingany or all of the features described above may be administered to thesubject via an adeno-associated viral (AAV) vector, such as an AAV5vector. The vector may be delivered to the retina of the subject (forexample, by subretinal injection). Various embodiments of the method maybe used in the treatment of human subjects. For example, the methods maybe used to treat subjects suffering from a CEP290 associated diseasesuch as LCA10, to restore CEP290 function in a subject in need thereof,and/or to alter a cell in the subject, such as a retinal cell and/or aphotoreceptor cell.

In another aspect, this disclosure relates to a nucleic acid encoding aCas9, a first gRNA with a targeting domain selected from SEQ ID NOs:389-391 (corresponding RNA sequences in SEQ ID NOs: 530, 468, and 538,respectively), and a second gRNA with a targeting domain selected fromSEQ ID NOs: 388, 392, and 394 (corresponding RNA sequences in SEQ IDNOs: 558, 460, and 568, respectively). The nucleic acid may, in variousembodiments, incorporate any or all of the features described above(e.g., the NLS and/or polyadenylation signal; the CMV, EFS or hGRK1promoter; and/or the ITRs). The nucleic acid may be part of an AAVvector, which vector may be used in medicine, for example to treat aCEP290 associated disease such as LCA10, and/or may be used to editspecific cells including retinal cells, for instance retinalphotoreceptor cells. The nucleic acid may also be used for theproduction of a medicament.

In yet another aspect, this disclosure relates to a method of treating asubject that includes the step of contacting a retina of the subjectwith one or more recombinant viral vectors (e.g., AAV vectors) thatencode a Cas9 and first and second gRNAs. The first and second gRNAs areadapted to form first and second ribonucleoprotein complexes with theCas9, and the first and second complexes in turn are adapted to cleavefirst and second target sequences, respectively, on either side of aCEP290 target position as that term is defined below. This cleavageresults in the alteration of the nucleic acid sequence of the CEP290target position. In some embodiments, the step of contacting the retinawith one or more recombinant viral vectors includes administering to theretina of the subject, by subretinal injection, a composition comprisingthe one or more recombinant viral vectors. The alteration of the nucleicacid sequence of the CEP290 target position can include formation of anindel, deletion of part or all of the CEP290 target position, and/orinversion of a nucleotide sequence in the CEP290 target position. Thesubject, in certain embodiments, is a primate.

The genome editing systems, compositions, and methods of the presentdisclosure can support high levels of productive editing in retinalcells, e.g., in photoreceptor cells. In certain embodiments, 10%, 15%,20%, or 25% of retinal cells in samples modified according to themethods of this disclosure (e.g., in retinal samples contacted with agenome editing system of this disclosure) comprise a productivealteration of an allele of the CEP290 gene. A productive alteration mayinclude, variously, a deletion and/or inversion of a sequence comprisingan IVS26 mutation, or another modification that results in an increasein the expression of functional CEP290 protein in a cell. In certainembodiments, 25%, 30%, 35%, 40%, 45%, 50%, or more than 50% ofphotoreceptor cells in retinal samples modified according to the methodsof this disclosure (e.g., in retinal samples contacted with a genomeediting system of this disclosure) comprise a productive alteration ofan allele of the CEP290 gene.

In another aspect, this disclosure relates to a nucleic acid encoding aCas9 and first and second gRNAs targeted to a CEP290 gene of a subjectfor use in therapy, e.g. in the treatment of CEP290-associated disease.The CEP290 associated disease may be, in some embodiments, LCA10, and inother embodiments may be selected from the group consisting ofSenior-Loken syndrome, Meckel Gruber syndrome, Bardet-Biedle syndromeand Joubert Syndrome. A targeting domain of the first gRNA may comprisea sequence selected from SEQ ID NOs: 389-391 (corresponding RNAsequences in SEQ ID NOs: 530, 468, and 538, respectively), and atargeting domain of the second gRNA may comprise a sequence selectedfrom SEQ ID NOs: 388, 392, and 394, respectively (corresponding RNAsequences in SEQ ID NOs: 558, 460, and 568, respectively). In certainembodiments, the first and second gRNA targeting domains comprise SEQ IDNOs: 389 and 388, respectively. In other embodiments, the first andsecond gRNA targeting domains comprise the sequences of SEQ ID NOs: 389and 392, respectively; SEQ ID NOs: 389 and 394, respectively; SEQ IDNOs: 390 and 388, respectively; SEQ ID NOs: 391 and 388, respectively;or SEQ ID NOs: 391 and 392, respectively. In still other embodiments,the first and second targeting domains comprise the sequences of SEQ IDNOs: 390 and 392, respectively; SEQ ID NOs: 390 and 394, respectively;or SEQ ID NOs: 391 and 394, respectively. The gRNAs according to thisaspect of the disclosure may be unimolecular, and may comprise RNAsequences according to SEQ ID NOs: 2779 or 2786 (corresponding to theDNA sequences of SEQ ID NOs: 2785 and 2787, respectively).Alternatively, the gRNAs may be two-part modular gRNAs according toeither sequence, where the crRNA component comprises the portion of SEQID NO: 2785/2779 or 2787/2786 that is underlined below, and the tracrRNAcomponent comprises the portion that is double-underlined below:

DNA: (SEQ ID NO: 2785)[N]₁₆₋₂₄GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTT and RNA: (SEQ ID NO: 2779)[N]₁₆₋₂₄GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUUU. DNA: (SEQ ID NO: 2787) N₁₆₋₂₄GTTATAGTACTCTGGAAACAGAATCTACTATAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTT and RNA: (SEQ ID NO: 2786)N₁₆₋₂₄GUUAUAGUACUCUGGAAACAGAAUCUACUAUAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUUU.

Continuing with this aspect of the disclosure, the Cas9 encoded by thenucleic acid is, in certain embodiments, a Staphylococcus aureus Cas9,which may be encoded by a sequence comprising SEQ ID NO: 39, or havingat least 80%, 85%, 90%, 95% or 99% sequence identity thereto. The Cas9encoded by the nucleic acid may comprise the amino acid sequence of SEQID NO: 26 or may share at least 80%, 85%, 90%, 95% or 99% sequenceidentity therewith. The Cas9 may be modified in some instances, forexample to include one or more nuclear localization signals (NLSs)(e.g., a C-terminal and an N-terminal NLS) and/or a polyadenylationsignal. Cas9 expression may be driven by a promoter sequence such as thepromoter sequence comprising SEQ ID NO: 401, the promoter sequencecomprising SEQ ID NO: 402, or the promoter sequence comprising SEQ IDNO: 403.

Staying with this aspect of the disclosure, the promoter sequence fordriving the expression of the Cas9 comprises, in certain embodiments,the sequence of a human GRK1 promoter. In other embodiments, thepromoter comprises the sequence of a cytomegalovirus (CMV) promoter oran EFS promoter. For example, the nucleic acid may comprise, in variousembodiments, (a) a CMV promoter for Cas9 and gRNAs comprising (ordiffering by no more than 3 nucleotides from) targeting domainsaccording to SEQ ID NOs: 389 and 392, or (b) a CMV promoter for Cas9 andgRNAs comprising targeting domains according to SEQ ID NOs: 389 and 394,or c) a CMV promoter for Cas9 and gRNAs comprising targeting domainsaccording to SEQ ID NOs: 390 and 388, or d) a CMV promoter for Cas9 andgRNAs comprising targeting domains according to SEQ ID NOs: 391 and 388,or e) a CMV promoter for Cas9 and gRNAs comprising targeting domainsaccording to SEQ ID NOs: 391 and 392, or f) an EFS promoter for Cas9 andgRNAs comprising targeting domains according to SEQ ID NOs: 389 and 392,or g) an EFS promoter for Cas9 and gRNAs comprising targeting domainsaccording to SEQ ID NOs: 389 and 394, or h) an EFS promoter for Cas9 andgRNAs comprising targeting domains according to SEQ ID NOs: 390 and 388,or i) an EFS promoter for Cas9 and gRNAs comprising targeting domainsaccording to SEQ ID NOs: 391 and 388, or j) an EFS promoter for Cas9 andgRNAs comprising targeting domains according to SEQ ID NOs: 391 and 392,or k) an hGRK1 promoter for Cas9 and gRNAs comprising targeting domainsaccording to SEQ ID NOs: 389 and 392, or g) an hGRK1 promoter for Cas9and gRNAs comprising targeting domains according to SEQ ID NOs: 389 and394, or h) an hGRK1 promoter for Cas9 and gRNAs comprising targetingdomains according to SEQ ID NOs: 390 and 388, or i) an hGRK1 promoterfor Cas9 and gRNAs comprising targeting domains according to SEQ ID NOs:391 and 388, or j) an hGRK1 promoter for Cas9 and gRNAs comprisingtargeting domains according to SEQ ID NOs: 391 and 392. In otherembodiments, the nucleic acid comprises a CMV promoter and guide RNAtargeting sequences according to SEQ ID NOs: 389 and 388. In still otherembodiments, the nucleic acid comprises an hGRK promoter and guide RNAtargeting sequences according to SEQ ID NOs: 390 and 392, or itcomprises a CMV promoter and guide RNA targeting sequences according toSEQ ID NOs: 390 and 392, or an hGRK promoter and guide RNA targetingsequences according to SEQ ID NOs: 390 and 394, or it comprises a CMVpromoter and guide RNA targeting sequences according to SEQ ID NOs: 391and 394, or an hGRK promoter and guide RNA targeting sequences accordingto SEQ ID NOs: 391 and 394, or it comprises a CMV promoter and guide RNAtargeting sequences according to SEQ ID NOs: 390 and 392. And in furtherembodiments, the promoter is hGRK or CMV while the first and second gRNAtargeting domains comprise the sequences of SEQ ID NOs: 389 and 392, SEQID NOs: 389 and 394, SEQ ID NOs: 390 and 388, SEQ ID NOs: 391 and 388,or SEQ ID NOs: 391 and 392.

In another aspect, the present disclosure relates to adeno-associatedvirus (AAV) vectors comprising the nucleic acids described above. AAVvectors comprising the foregoing nucleic acids may be administered to avariety of tissues of a subject, though in certain embodiments the AAVvectors are administered to a retina of the subject, and/or areadministered by subretinal injection. The AAV vector may comprise anAAV5 capsid.

An additional aspect of this disclosure relates to a nucleic acid asdescribed above, for delivery via an AAV vector also as described above.The nucleic acid includes in some embodiments, first and second invertedterminal repeat sequences (ITRs), a first guide RNA comprising atargeting domain sequence selected from SEQ ID NOs: 389-391(corresponding RNA sequences in SEQ ID NOs: 530, 468, and 538,respectively), a second guide RNA comprising a targeting domain sequenceselected from SEQ ID NOs: 388, 392, and 394 (corresponding RNA sequencesin SEQ ID NOs: 558, 460, and 568, respectively), and a promoter fordriving Cas9 expression comprising a sequence selected from SEQ ID NOs:401-403. In certain embodiments, the nucleic acid includes first andsecond ITRs and first and second guide RNAs comprising a guide RNAsequence selected from SEQ ID NOs: 2785 and 2787 (e.g., both first andsecond guide RNAs comprise the sequence of SEQ ID NO: 2787). The nucleicacid may be used in the treatment of human subjects, and/or in theproduction of a medicament.

The nucleic acids and vectors according to these aspects of thedisclosure may be used in medicine, for instance in the treatment ofdisease. In some embodiments, they are used in the treatment of aCEP290-associated disease, in the treatment of LCA10, or in thetreatment of one or more of the following: Senior-Loken syndrome, MeckelGruber syndrome, Bardet-Biedle syndrome, and/or Joubert Syndrome.Without wishing to be bound by theory, it is contemplated that thenucleic acids and vectors disclosed herein may be used to treat otherinherited retinal diseases by adapting the gRNA targeting domains totarget and alter the gene of interest. In certain embodiments, thenucleic acids and vectors according to the disclosure may be used forthe treatment of other inherited retinal diseases as set forth in Stone2017, which is incorporated by reference herein in its entirety. Forexample, in certain embodiments, the nucleic acids and vectors disclosedherein may be used to treat USH2A-related disorders by including gRNAscomprising targeting domains that alter the USH2A gene. Vectors andnucleic acids according to this disclosure may be administered to theretina of a subject, for instance by subretinal injection.

This disclosure also relates to recombinant viral vectors comprising thenucleic acids described above, and to the use of such viral vectors inthe treatment of disease. In some embodiments, one or more viral vectorsencodes a Cas9, a first gRNA and a second gRNA for use in a method ofaltering a nucleotide sequence of a CEP 290 target position wherein (a)the first and second gRNAs are adapted to form first and secondribonucleoprotein complexes with the Cas9, and (b) the first and secondribonucleoprotein complexes are adapted to cleave first and secondcellular nucleic acid sequences on first and second sides of a CEP290target position, thereby altering a nucleotide sequence of the CEP290target position. In use, the one or more recombinant viral vectors iscontacted to the retina of a subject, for instance by subretinalinjection.

Another aspect of this disclosure relates to AAV vectors, AAV vectorgenomes and/or nucleic acids that may be carried by AAV vectors, whichencode one or more guide RNAs, each comprising a sequence selectedfrom—or having at least 90% sequence identity to—one of SEQ ID NOs: 2785or 2787, a sequence encoding a Cas9 and a promoter sequence operablycoupled to the Cas9 coding sequence, which promoter sequence comprises asequence selected from—or having at least 90% sequence identity to—oneof SEQ ID NOs: 401-403. The Cas9 coding sequence may comprise thesequence of SEQ ID NO: 39, or it may share at least 90% sequenceidentity therewith. Alternatively or additionally, the Cas9 codingsequence may encode an amino acid sequence comprising SEQ ID NO: 26, orsharing at least 90% sequence identity therewith. In certainembodiments, the AAV vector, vector genome or nucleic acid furthercomprises one or more of the following: left and right ITR sequences,optionally selected from—or having at least 90% sequence identity to—SEQID NOs: 408 and 437, respectively; and one or more U6 promoter sequencesoperably coupled to the one or more guide RNA sequences. The U6 promotersequences may comprise, or share at least 90% sequence identity with,SEQ ID NO: 417.

Methods and compositions discussed herein, provide for treating ordelaying the onset or progression of diseases of the eye, e.g.,disorders that affect retinal cells, e.g., photoreceptor cells.

Methods and compositions discussed herein, provide for treating ordelaying the onset or progression of Leber's Congenital Amaurosis 10(LCA10), an inherited retinal degenerative disease characterized bysevere loss of vision at birth. LCA10 is caused by a mutation in theCEP290 gene, e.g., a c.2991+1655A to G (adenine to guanine) mutation inthe CEP290 gene which gives rise to a cryptic splice site in intron 26.This is a mutation at nucleotide 1655 of intron 26 of CEP290, e.g., an Ato G mutation. CEP290 is also known as: CT87; MKS4; POC3; rd16; BBS14;JBTS5; LCA10; NPHP6; SLSN6; and 3H11Ag.

Methods and compositions discussed herein, provide for treating ordelaying the onset or progression of LCA10 by gene editing, e.g., usingCRISPR-Cas9 mediated methods to alter a LCA10 target position, asdisclosed below.

“LCA10 target position” as used herein refers to nucleotide 1655 ofintron 26 of the CEP290 gene, and the mutation at that site that givesrise to a cryptic splice donor site in intron 26 which results in theinclusion of an aberrant exon of 128 bp (c.2991+1523 to c.2991+1650) inthe mutant CEP290 mRNA, and inserts a premature stop codon (p.C998X).The sequence of the cryptic exon contains part of an Alu repeat region.The Alu repeats span from c.2991+1162 to c.2991+1638. In an embodiment,the LCA10 target position is occupied by an adenine (A) to guanine (G)mutation (c.2991+1655A to G).

In one aspect, methods and compositions discussed herein, provide foraltering a LCA10 target position in the CEP290 gene. The methods andcompositions described herein introduce one or more breaks near the siteof the LCA target position (e.g., c.2991+1655A to G) in at least oneallele of the CEP290 gene. Altering the LCA10 target position refers to(1) break-induced introduction of an indel (also referred to herein asNHEJ-mediated introduction of an indel) in close proximity to orincluding a LCA10 target position (e.g., c.2991+1655A to G), or (2)break-induced deletion (also referred to herein as NHEJ-mediateddeletion) of genomic sequence including the mutation at a LCA10 targetposition (e.g., c.2991+1655A to G). Both approaches give rise to theloss or destruction of the cryptic splice site resulting from themutation at the LCA10 target position (e.g., c.2991+1655A to G).

In an embodiment, a single strand break is introduced in close proximityto or at the LCA10 target position (e.g., c.2991+1655A to G) in theCEP290 gene. While not wishing to be bound by theory, it is believedthat break-induced indels (e.g., indels created following NHEJ) destroythe cryptic splice site. In an embodiment, the single strand break willbe accompanied by an additional single strand break, positioned by asecond gRNA molecule.

In an embodiment, a double strand break is introduced in close proximityto or at the LCA10 target position (e.g., c.2991+1655A to G) in theCEP290 gene. While not wishing to be bound by theory, it is believedthat break-induced indels (e.g., indels created following NHEJ) destroythe cryptic splice site. In an embodiment, a double strand break will beaccompanied by an additional single strand break may be positioned by asecond gRNA molecule. In an embodiment, a double strand break will beaccompanied by two additional single strand breaks positioned by asecond gRNA molecule and a third gRNA molecule.

In an embodiment, a pair of single strand breaks is introduced in closeproximity to or at the LCA10 target position (e.g., c.2991+1655A to G)in the CEP290 gene. While not wishing to be bound by theory, it isbelieved that break-induced indels destroy the cryptic splice site. Inan embodiment, the pair of single strand breaks will be accompanied byan additional double strand break, positioned by a third gRNA molecule.In an embodiment, the pair of single strand breaks will be accompaniedby an additional pair of single strand breaks positioned by a third gRNAmolecule and a fourth gRNA molecule.

In an embodiment, two double strand breaks are introduced to flank theLCA10 target position in the CEP290 gene (one 5′ and the other one 3′ tothe mutation at the LCA10 target position, e.g., c.2991+1655A to G) toremove (e.g., delete) the genomic sequence including the mutation at theLCA10 target position. It is contemplated herein that in an embodimentthe break-induced deletion of the genomic sequence including themutation at the LCA10 target position is mediated by NHEJ. In anembodiment, the breaks (i.e., the two double strand breaks) arepositioned to avoid unwanted target chromosome elements, such as repeatelements, e.g., an Alu repeat. The breaks, i.e., two double strandbreaks, can be positioned upstream and downstream of the LCA10 targetposition, as discussed herein.

In an embodiment, one double strand break (either 5′ or 3′ to themutation at the LCA10 target position, e.g., c.2991+1655A to G) and twosingle strand breaks (on the other side of the mutation at the LCA10target position from the double strand break) are introduced to flankthe LCA10 target position in the CEP290 gene to remove (e.g., delete)the genomic sequence including the mutation at the LCA10 targetposition. It is contemplated herein that in an embodiment thebreak-induced deletion of the genomic sequence including the mutation atthe LCA10 target position is mediated by NHEJ. In an embodiment, thebreaks (i.e., the double strand break and the two single strand breaks)are positioned to avoid unwanted target chromosome elements, such asrepeat elements, e.g., an Alu repeat. The breaks, e.g., one doublestrand break and two single strand breaks, can be positioned upstreamand downstream of the LCA10 target position, as discussed herein.

In an embodiment, two pairs of single strand breaks (two 5′ and theother two 3′ to the mutation at the LCA10 target position, e.g.,c.2991+1655A to G) are introduced to flank the LCA10 target position inthe CEP290 gene to remove (e.g., delete) the genomic sequence includingthe mutation at the LCA10 target position. It is contemplated hereinthat in an embodiment the break-induced deletion of the genomic sequenceincluding the mutation at the LCA10 target position is mediated by NHEJ.In an embodiment, the breaks (e.g., two pairs of single strand breaks)are positioned to avoid unwanted target chromosome elements, such asrepeat elements, e.g., an Alu repeat. The breaks, e.g., two pairs ofsingle strand breaks, can be positioned upstream or downstream of theLCA10 target position, as discussed herein.

The LCA10 target position may be targeted by cleaving with either asingle nuclease or dual nickases, e.g., to induce break-induced indel inclose proximity to or including the LCA10 target position orbreak-induced deletion of genomic sequence including the mutation at theLCA10 target position in the CEP290 gene. The method can includeacquiring knowledge of the mutation carried by the subject, e.g., bysequencing the appropriate portion of the CEP290 gene.

In one aspect, disclosed herein is a gRNA molecule, e.g., an isolated ornon-naturally occurring gRNA molecule, comprising a targeting domainwhich is complementary with a target domain from the CEP290 gene.

When two or more gRNAs are used to position two or more cleavage events,e.g., double strand or single strand breaks, in a target nucleic acid,it is contemplated that in an embodiment the two or more cleavage eventsmay be made by the same or different Cas9 proteins. For example, whentwo gRNAs are used to position two double strand breaks, a single Cas9nuclease may be used to create both double strand breaks. When two ormore gRNAs are used to position two or more single stranded breaks(single strand breaks), a single Cas9 nickase may be used to create thetwo or more single strand breaks. When two or more gRNAs are used toposition at least one double strand break and at least one single strandbreak, two Cas9 proteins may be used, e.g., one Cas9 nuclease and oneCas9 nickase. It is contemplated that in an embodiment when two or moreCas9 proteins are used that the two or more Cas9 proteins may bedelivered sequentially to control specificity of a double strand versusa single strand break at the desired position in the target nucleicacid.

In some embodiments, the targeting domain of the first gRNA molecule andthe targeting domain of the second gRNA molecule hybridize to the targetdomain from the target nucleic acid molecule (i.e., the CEP290 gene)through complementary base pairing to opposite strands of the targetnucleic acid molecule. In some embodiments, the first gRNA molecule andthe second gRNA molecule are configured such that the PAMs are orientedoutward.

In an embodiment, the targeting domain of a gRNA molecule is configuredto avoid unwanted target chromosome elements, such as repeat elements,e.g., an Alu repeat, or the endogenous CEP290 splice sites, in thetarget domain. The gRNA molecule may be a first, second, third and/orfourth gRNA molecule.

In an embodiment, the targeting domain of a gRNA molecule is configuredto position a cleavage event sufficiently far from a preselectednucleotide, e.g., the nucleotide of a coding region, such that thenucleotide is not altered. In an embodiment, the targeting domain of agRNA molecule is configured to position an intronic cleavage eventsufficiently far from an intron/exon border, or naturally occurringsplice signal, to avoid alteration of the exonic sequence or unwantedsplicing events. The gRNA molecule may be a first, second, third and/orfourth gRNA molecule, as described herein.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Table 11. In someembodiments, the targeting domain is selected from those in Table 11.For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 387) GACACTGCCAATAGGGATAGGT; (SEQ ID NO: 388)GTCAAAAGCTACCGGTTACCTG; (SEQ ID NO: 389) GTTCTGTCCTCAGTAAAAGGTA;(SEQ ID NO: 390) GAATAGTTTGTTCTGGGTAC; (SEQ ID NO: 391)GAGAAAGGGATGGGCACTTA; (SEQ ID NO: 392) GATGCAGAACTAGTGTAGAC;(SEQ ID NO: 393) GTCACATGGGAGTCACAGGG; or (SEQ ID NO: 394)GAGTATCTCCTGTTTGGCA.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Table 11. In an embodiment, the two or more gRNAs or targetingdomains are selected from one or more of the pairs of gRNAs or targetingdomains described herein, e.g., as indicated in Table 11. In anembodiment, when two or more gRNAs are used to position four breaks,e.g., four single strand breaks in the target nucleic acid sequence,each guide RNA is independently selected from one of Table 11.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Table 2A-2D. In someembodiments, the targeting domain is selected from those in Table 2A-2D.For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 395) GAGAUACUCACAAUUACAAC; or (SEQ ID NO: 396)GAUACUCACAAUUACAACUG.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 2A-2D. In an embodiment, when two or more gRNAs are used toposition four breaks, e.g., four single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 2A-2D.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Tables 3A-3C. In someembodiments, the targeting domain is selected from those in Tables3A-3C. For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 395) GAGAUACUCACAAUUACAAC; or (SEQ ID NO: 397)GAUACUCACAAUUACAA.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single stranded breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 3A-3C. In an embodiment, when two or more gRNAs are used toposition four breaks, e.g., four single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 3A-3C.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Tables 7A-7D. In someembodiments, the targeting domain is selected from those in Tables7A-7D. For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 398) GCACCUGGCCCCAGUUGUAAUU.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single stranded breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 7A-7D. In an embodiment, when two or more gRNAs are used toposition four breaks, e.g., four single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 7A-7D.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Tables 4A-4D. In someembodiments, the targeting domain is selected from those in Tables4A-4D. For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 457) GCUACCGGUUACCUGAA; (SEQ ID NO: 458) GCAGAACUAGUGUAGAC;(SEQ ID NO: 459) GUUGAGUAUCUCCUGUU; (SEQ ID NO: 460)GAUGCAGAACUAGUGUAGAC; or (SEQ ID NO: 461) GCUUGAACUCUGUGCCAAAC.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single stranded breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 4A-4D. In an embodiment, when two or more gRNAs are used toposition four breaks, e.g., four single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 4A-4D.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Tables 8A-8D. In someembodiments, the targeting domain is selected from those in Tables8A-8D. For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 457) GCUACCGGUUACCUGAA; (SEQ ID NO: 458) GCAGAACUAGUGUAGAC;(SEQ ID NO: 459) GUUGAGUAUCUCCUGUU; (SEQ ID NO: 460)GAUGCAGAACUAGUGUAGAC; (SEQ ID NO: 461) GCUUGAACUCUGUGCCAAAC; (SEQ ID NO:462) GAAAGAUGAAAAAUACUCUU; (SEQ ID NO: 463) GAAAUAGAUGUAGAUUG; (SEQ IDNO: 464) GAAAUAUUAAGGGCUCUUCC; (SEQ ID NO: 465) GAACAAAAGCCAGGGACCAU;(SEQ ID NO: 466) GAACUCUAUACCUUUUACUG; (SEQ ID NO: 467)GAAGAAUGGAAUAGAUAAUA; (SEQ ID NO: 468) GAAUAGUUUGUUCUGGGUAC; (SEQ ID NO:469) GAAUGGAAUAGAUAAUA; (SEQ ID NO: 470) GAAUUUACAGAGUGCAUCCA; (SEQ IDNO: 471) GAGAAAAAGGAGCAUGAAAC; (SEQ ID NO: 472) GAGAGCCACAGUGCAUG; (SEQID NO: 473) GAGGUAGAAUCAAGAAG; (SEQ ID NO: 474) GAGUGCAUCCAUGGUCC; (SEQID NO: 475) GAUAACUACAAAGGGUC; (SEQ ID NO: 476) GAUAGAGACAGGAAUAA; (SEQID NO: 477) GAUGAAAAAUACUCUUU; (SEQ ID NO: 478) GAUGACAUGAGGUAAGU; (SEQID NO: 479) GCAUGUGGUGUCAAAUA; (SEQ ID NO: 480) GCCUGAACAAGUUUUGAAAC;(SEQ ID NO: 481) GCUCUUUUCUAUAUAUA; (SEQ ID NO: 482)GCUUUUGACAGUUUUUAAGG; (SEQ ID NO: 483) GCUUUUGUUCCUUGGAA; (SEQ ID NO:484) GGAACAAAAGCCAGGGACCA; (SEQ ID NO: 485) GGACUUGACUUUUACCCUUC; (SEQID NO: 486) GGAGAAUAGUUUGUUCU; (SEQ ID NO: 487) GGAGUCACAUGGGAGUCACA;(SEQ ID NO: 488) GGAUAGGACAGAGGACA; (SEQ ID NO: 489)GGCUGUAAGAUAACUACAAA; (SEQ ID NO: 490) GGGAGAAUAGUUUGUUC; (SEQ ID NO:491) GGGAGUCACAUGGGAGUCAC; (SEQ ID NO: 492) GGGCUCUUCCUGGACCA; (SEQ IDNO: 493) GGGUACAGGGGUAAGAGAAA; (SEQ ID NO: 494) GGUCCCUGGCUUUUGUUCCU;(SEQ ID NO: 495) GUAAAGGUUCAUGAGACUAG; (SEQ ID NO: 496)GUAACAUAAUCACCUCUCUU; (SEQ ID NO: 497) GUAAGACUGGAGAUAGAGAC; (SEQ ID NO:498) GUACAGGGGUAAGAGAA; (SEQ ID NO: 499) GUAGCUUUUGACAGUUUUUA; (SEQ IDNO: 500) GUCACAUGGGAGUCACA; (SEQ ID NO: 501) GUGGAGAGCCACAGUGCAUG; (SEQID NO: 502) GUUACAAUCUGUGAAUA; (SEQ ID NO: 503) GUUCUGUCCUCAGUAAA; (SEQID NO: 504) GUUUAGAAUGAUCAUUCUUG; (SEQ ID NO: 505) GUUUGUUCUGGGUACAG;(SEQ ID NO: 506) UAAAAACUGUCAAAAGCUAC; (SEQ ID NO: 507)UAAAAGGUAUAGAGUUCAAG; (SEQ ID NO: 508) UAAAUCAUGCAAGUGACCUA; or (SEQ IDNO: 509) UAAGAUAACUACAAAGGGUC.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single stranded breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 8A-8D. In an embodiment, when two or more gRNAs are used toposition four breaks, e.g., four single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 8A-8D.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Table 5A-5D. In someembodiments, the targeting domain is selected from those in Table 5A-5D.For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 510) GAAUCCUGAAAGCUACU.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single stranded breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 5A-5D. In an embodiment, when two or more gRNAs are used toposition four breaks, e.g., four single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 5A-5D.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Tables 9A-9E. In someembodiments, the targeting domain is selected from those in Tables9A-9E. For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 460) GAUGCAGAACUAGUGUAGAC; (SEQ ID NO: 468)GAAUAGUUUGUUCUGGGUAC; (SEQ ID NO: 480) GCCUGAACAAGUUUUGAAAC; (SEQ ID NO:494) GGUCCCUGGCUUUUGUUCCU; (SEQ ID NO: 497) GUAAGACUGGAGAUAGAGAC; (SEQID NO: 511) GCUAAAUCAUGCAAGUGACCUAAG; (SEQ ID NO: 512)GGUCACUUGCAUGAUUUAG; (SEQ ID NO: 513) GUCACUUGCAUGAUUUAG; (SEQ ID NO:514) GCCUAGGACUUUCUAAUGCUGGA; (SEQ ID NO: 515) GGACUUUCUAAUGCUGGA; (SEQID NO: 516) GGGACCAUGGGAGAAUAGUUUGUU; (SEQ ID NO: 517)GGACCAUGGGAGAAUAGUUUGUU; (SEQ ID NO: 518) GACCAUGGGAGAAUAGUUUGUU; (SEQID NO: 519) GGUCCCUGGCUUUUGUUCCUUGGA; (SEQ ID NO: 520)GUCCCUGGCUUUUGUUCCUUGGA; (SEQ ID NO: 521) GAAAACGUUGUUCUGAGUAGCUUU; (SEQID NO: 522) GUUGUUCUGAGUAGCUUU; (SEQ ID NO: 523) GUCCCUGGCUUUUGUUCCU;(SEQ ID NO: 524) GACAUCUUGUGGAUAAUGUAUCA; (SEQ ID NO: 525)GUCCUAGGCAAGAGACAUCUU; (SEQ ID NO: 526) GCCAGCAAAAGCUUUUGAGCUAA; (SEQ IDNO: 527) GCAAAAGCUUUUGAGCUAA; (SEQ ID NO: 528) GAUCUUAUUCUACUCCUGUGA;(SEQ ID NO: 529) GCUUUCAGGAUUCCUACUAAAUU; (SEQ ID NO: 530)GUUCUGUCCUCAGUAAAAGGUA; (SEQ ID NO: 531) GAACAACGUUUUCAUUUA; (SEQ ID NO:532) GUAGAAUAUCAUAAGUUACAAUCU; (SEQ ID NO: 533) GAAUAUCAUAAGUUACAAUCU;(SEQ ID NO: 534) GUGGCUGUAAGAUAACUACA; (SEQ ID NO: 535)GGCUGUAAGAUAACUACA; (SEQ ID NO: 536) GUUUAACGUUAUCAUUUUCCCA; (SEQ ID NO:537) GUAAGAGAAAGGGAUGGGCACUUA; (SEQ ID NO: 538) GAGAAAGGGAUGGGCACUUA;(SEQ ID NO: 539) GAAAGGGAUGGGCACUUA; (SEQ ID NO: 540)GUAAAUGAAAACGUUGUU; (SEQ ID NO: 541) GAUAAACAUGACUCAUAAUUUAGU; (SEQ IDNO: 542) GGAACAAAAGCCAGGGACCAUGG; (SEQ ID NO: 543)GAACAAAAGCCAGGGACCAUGG; (SEQ ID NO: 544) GGGAGAAUAGUUUGUUCUGGGUAC; (SEQID NO: 545) GGAGAAUAGUUUGUUCUGGGUAC; (SEQ ID NO: 546)GAGAAUAGUUUGUUCUGGGUAC; (SEQ ID NO: 547) GAAAUAGAGGCUUAUGGAUU; (SEQ IDNO: 548) GUUCUGGGUACAGGGGUAAGAGAA; (SEQ ID NO: 549) GGGUACAGGGGUAAGAGAA;(SEQ ID NO: 550) GGUACAGGGGUAAGAGAA; (SEQ ID NO: 551)GUAAAUUCUCAUCAUUUUUUAUUG; (SEQ ID NO: 552) GGAGAGGAUAGGACAGAGGACAUG;(SEQ ID NO: 553) GAGAGGAUAGGACAGAGGACAUG; (SEQ ID NO: 554)GAGGAUAGGACAGAGGACAUG; (SEQ ID NO: 555) GGAUAGGACAGAGGACAUG; (SEQ ID NO:556) GAUAGGACAGAGGACAUG; (SEQ ID NO: 557) GAAUAAAUGUAGAAUUUUAAUG; (SEQID NO: 558) GUCAAAAGCUACCGGUUACCUG; (SEQ ID NO: 559)GUUUUUAAGGCGGGGAGUCACAU; (SEQ ID NO: 560) GUCUUACAUCCUCCUUACUGCCAC; (SEQID NO: 561) GAGUCACAGGGUAGGAUUCAUGUU; (SEQ ID NO: 562)GUCACAGGGUAGGAUUCAUGUU; (SEQ ID NO: 563) GGCACAGAGUUCAAGCUAAUACAU; (SEQID NO: 564) GCACAGAGUUCAAGCUAAUACAU; (SEQ ID NO: 565)GAGUUCAAGCUAAUACAU; (SEQ ID NO: 566) GUGUUGAGUAUCUCCUGUUUGGCA; (SEQ IDNO: 567) GUUGAGUAUCUCCUGUUUGGCA; (SEQ ID NO: 568) GAGUAUCUCCUGUUUGGCA;(SEQ ID NO: 569) GAAAAUCAGAUUUCAUGUGUG; (SEQ ID NO: 570)GCCACAAGAAUGAUCAUUCUAAAC; (SEQ ID NO: 571) GGCGGGGAGUCACAUGGGAGUCA; (SEQID NO: 572) GCGGGGAGUCACAUGGGAGUCA; (SEQ ID NO: 573)GGGGAGUCACAUGGGAGUCA; (SEQ ID NO: 574) GGGAGUCACAUGGGAGUCA; (SEQ ID NO:575) GGAGUCACAUGGGAGUCA; (SEQ ID NO: 576) GCUUUUGACAGUUUUUAAGGCG; (SEQID NO: 577) GAUCAUUCUUGUGGCAGUAAG; (SEQ ID NO: 578) GAGCAAGAGAUGAACUAG;(SEQ ID NO: 579) GUAGAUUGAGGUAGAAUCAAGAA; (SEQ ID NO: 580)GAUUGAGGUAGAAUCAAGAA; (SEQ ID NO: 581) GGAUGUAAGACUGGAGAUAGAGAC; (SEQ IDNO: 582) GAUGUAAGACUGGAGAUAGAGAC; (SEQ ID NO: 583)GGGAGUCACAUGGGAGUCACAGGG; (SEQ ID NO: 584) GGAGUCACAUGGGAGUCACAGGG; (SEQID NO: 585) GAGUCACAUGGGAGUCACAGGG; (SEQ ID NO: 586)GUCACAUGGGAGUCACAGGG; (SEQ ID NO: 587) GUUUACAUAUCUGUCUUCCUUAA; or (SEQID NO: 588) GAUUUCAUGUGUGAAGAA.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single stranded breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 9A-9E. In an embodiment, when two or more gRNAs are used toposition four breaks, e.g., four single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 9A-9E.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Tables 6A-6B. In someembodiments, the targeting domain is selected from those in Tables6A-6B. For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 589) GAGUUCAAGCUAAUACAUGA; (SEQ ID NO: 590)GUUGUUCUGAGUAGCUU; or (SEQ ID NO: 591) GGCAAAAGCAGCAGAAAGCA.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single stranded breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 6A-6B. In an embodiment, when two or more gRNAs are used toposition four breaks, e.g., four single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 6A-6B.

In an embodiment, the LCA10 target position in the CEP290 gene istargeted. In an embodiment, the targeting domain comprises a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from Tables 10A-10B. Insome embodiments, the targeting domain is selected from those in Tables10A-10B. For example, in certain embodiments, the targeting domain is:

(SEQ ID NO: 589) GAGUUCAAGCUAAUACAUGA; (SEQ ID NO: 590)GUUGUUCUGAGUAGCUU; (SEQ ID NO: 591) GGCAAAAGCAGCAGAAAGCA; (SEQ ID NO:592) GUGGCUGAAUGACUUCU; or (SEQ ID NO: 593) GACUAGAGGUCACGAAA.

In an embodiment, when two or more gRNAs are used to position two ormore breaks, e.g., two or more single stranded breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 10A-10B. In an embodiment, when two or more gRNAs are used toposition four breaks, e.g., four single strand breaks in the targetnucleic acid sequence, each guide RNA is independently selected from oneof Tables 10A-10B.

In an embodiment, the gRNA, e.g., a gRNA comprising a targeting domain,which is complementary with a target domain from the CEP290 gene, is amodular gRNA. In other embodiments, the gRNA is a chimeric gRNA.

In an embodiment, when two gRNAs are used to position two breaks, e.g.,two single strand breaks, in the target nucleic acid sequence, eachguide RNA is independently selected from one or more of Tables 2A-2D,Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D,Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11.

In an embodiment, the targeting domain which is complementary with atarget domain from the CEP290 gene comprises 16 or more nucleotides inlength. In an embodiment, the targeting domain which is complementarywith a target domain from the CEP290 gene is 16 nucleotides or more inlength. In an embodiment, the targeting domain is 16 nucleotides inlength. In an embodiment, the targeting domain is 17 nucleotides inlength. In an embodiment, the targeting domain is 18 nucleotides inlength. In an embodiment, the targeting domain is 19 nucleotides inlength. In an embodiment, the targeting domain is 20 nucleotides inlength. In an embodiment, the targeting domain is 21 nucleotides inlength. In an embodiment, the targeting domain is 22 nucleotides inlength. In an embodiment, the targeting domain is 23 nucleotides inlength. In an embodiment, the targeting domain is 24 nucleotides inlength. In an embodiment, the targeting domain is 25 nucleotides inlength. In an embodiment, the targeting domain is 26 nucleotides inlength.

In an embodiment, the targeting domain comprises 16 nucleotides.

In an embodiment, the targeting domain comprises 17 nucleotides.

In an embodiment, the targeting domain comprises 18 nucleotides.

In an embodiment, the targeting domain comprises 19 nucleotides.

In an embodiment, the targeting domain comprises 20 nucleotides.

In an embodiment, the targeting domain comprises 21 nucleotides.

In an embodiment, the targeting domain comprises 22 nucleotides.

In an embodiment, the targeting domain comprises 23 nucleotides.

In an embodiment, the targeting domain comprises 24 nucleotides.

In an embodiment, the targeting domain comprises 25 nucleotides.

In an embodiment, the targeting domain comprises 26 nucleotides.

A gRNA as described herein may comprise from 5′ to 3′: a targetingdomain (comprising a “core domain”, and optionally a “secondarydomain”); a first complementarity domain; a linking domain; a secondcomplementarity domain; a proximal domain; and a tail domain. In someembodiments, the proximal domain and tail domain are taken together as asingle domain.

In an embodiment, a gRNA comprises a linking domain of no more than 25nucleotides in length; a proximal and tail domain, that taken together,are at least 20 nucleotides in length; and a targeting domain of equalto or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

In another embodiment, a gRNA comprises a linking domain of no more than25 nucleotides in length; a proximal and tail domain, that takentogether, are at least 30 nucleotides in length; and a targeting domainof equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

In another embodiment, a gRNA comprises a linking domain of no more than25 nucleotides in length; a proximal and tail domain, that takentogether, are at least 30 nucleotides in length; and a targeting domainof equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

In another embodiment, a gRNA comprises a linking domain of no more than25 nucleotides in length; a proximal and tail domain, that takentogether, are at least 40 nucleotides in length; and a targeting domainof equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

A cleavage event, e.g., a double strand or single strand break, isgenerated by a Cas9 molecule. The Cas9 molecule may be an enzymaticallyactive Cas9 (eaCas9) molecule, e.g., an eaCas9 molecule that forms adouble strand break in a target nucleic acid or an eaCas9 molecule formsa single strand break in a target nucleic acid (e.g., a nickasemolecule).

In an embodiment, the eaCas9 molecule catalyzes a double strand break.

In some embodiments, the eaCas9 molecule comprises HNH-like domaincleavage activity but has no, or no significant, N-terminal RuvC-likedomain cleavage activity. In this case, the eaCas9 molecule is anHNH-like domain nickase, e.g., the eaCas9 molecule comprises a mutationat D10, e.g., D10A. In other embodiments, the eaCas9 molecule comprisesN-terminal RuvC-like domain cleavage activity but has no, or nosignificant, HNH-like domain cleavage activity. In an embodiment, theeaCas9 molecule is an N-terminal RuvC-like domain nickase, e.g., theeaCas9 molecule comprises a mutation at H840, e.g., H840A. In anembodiment, the eaCas9 molecule is an N-terminal RuvC-like domainnickase, e.g., the eaCas9 molecule comprises a mutation at H863, e.g.,H863A.

In an embodiment, a single strand break is formed in the strand of thetarget nucleic acid to which the targeting domain of said gRNA iscomplementary. In another embodiment, a single strand break is formed inthe strand of the target nucleic acid other than the strand to which thetargeting domain of said gRNA is complementary.

In another aspect, disclosed herein is a nucleic acid, e.g., an isolatedor non-naturally occurring nucleic acid, e.g., DNA, that comprises (a) asequence that encodes a gRNA molecule comprising a targeting domain thatis complementary with a target domain in CEP290 gene as disclosedherein.

In an embodiment, the nucleic acid encodes a gRNA molecule, e.g., thefirst gRNA molecule, comprising a targeting domain comprising a sequencethat is the same as, or differs by no more than 1, 2, 3, 4, or 5nucleotides from, a targeting domain sequence from any one of Tables2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11. In anembodiment, the nucleic acid encodes a gRNA molecule comprising atargeting domain that is selected from those in Tables 2A-2D, Tables3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11.

In an embodiment, the nucleic acid encodes a modular gRNA, e.g., one ormore nucleic acids encode a modular gRNA. In other embodiments, thenucleic acid encodes a chimeric gRNA. The nucleic acid may encode agRNA, e.g., the first gRNA molecule, comprising a targeting domaincomprising 16 nucleotides or more in length. In one embodiment, thenucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising atargeting domain that is 16 nucleotides in length. In other embodiments,the nucleic acid encodes a gRNA, e.g., the first gRNA molecule,comprising a targeting domain that is 17 nucleotides in length. In stillother embodiments, the nucleic acid encodes a gRNA, e.g., the first gRNAmolecule, comprising a targeting domain that is 18 nucleotides inlength. In still other embodiments, the nucleic acid encodes a gRNA,e.g., the first gRNA molecule, comprising a targeting domain that is 19nucleotides in length. In still other embodiments, the nucleic acidencodes a gRNA, e.g., the first gRNA molecule, comprising a targetingdomain that is 20 nucleotides in length. In still other embodiments, thenucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising atargeting domain that is 21 nucleotides in length. In still otherembodiments, the nucleic acid encodes a gRNA, e.g., the first gRNAmolecule, comprising a targeting domain that is 22 nucleotides inlength. In still other embodiments, the nucleic acid encodes a gRNA,e.g., the first gRNA molecule, comprising a targeting domain that is 23nucleotides in length. In still other embodiments, the nucleic acidencodes a gRNA, e.g., the first gRNA molecule, comprising a targetingdomain that is 24 nucleotides in length. In still other embodiments, thenucleic acid encodes a gRNA, e.g., the first gRNA molecule, comprising atargeting domain that is 25 nucleotides in length. In still otherembodiments, the nucleic acid encodes a gRNA, e.g., the first gRNAmolecule, comprising a targeting domain that is 26 nucleotides inlength.

In an embodiment, a nucleic acid encodes a gRNA comprising from 5′ to3′: a targeting domain (comprising a “core domain”, and optionally a“secondary domain”); a first complementarity domain; a linking domain; asecond complementarity domain; a proximal domain; and a tail domain. Insome embodiments, the proximal domain and tail domain are taken togetheras a single domain.

In an embodiment, a nucleic acid encodes a gRNA e.g., the first gRNAmolecule, comprising a linking domain of no more than 25 nucleotides inlength; a proximal and tail domain, that taken together, are at least 20nucleotides in length; and a targeting domain of equal to or greaterthan 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a gRNA e.g., the first gRNAmolecule, comprising a linking domain of no more than 25 nucleotides inlength; a proximal and tail domain, that taken together, are at least 30nucleotides in length; and a targeting domain of equal to or greaterthan 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a gRNA e.g., the first gRNAmolecule, comprising a linking domain of no more than 25 nucleotides inlength; a proximal and tail domain, that taken together, are at least 30nucleotides in length; and a targeting domain of equal to or greaterthan 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid encodes a gRNA comprising e.g., thefirst gRNA molecule, a linking domain of no more than 25 nucleotides inlength; a proximal and tail domain, that taken together, are at least 40nucleotides in length; and a targeting domain of equal to or greaterthan 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, a nucleic acid comprises (a) a sequence that encodes agRNA molecule comprising a targeting domain that is complementary with atarget domain in the CEP290 gene as disclosed herein, and furthercomprises (b) a sequence that encodes a Cas9 molecule.

The Cas9 molecule may be a nickase molecule, a enzymatically activatingCas9 (eaCas9) molecule, e.g., an eaCas9 molecule that forms a doublestrand break in a target nucleic acid and an eaCas9 molecule forms asingle strand break in a target nucleic acid. In an embodiment, a singlestrand break is formed in the strand of the target nucleic acid to whichthe targeting domain of said gRNA is complementary. In anotherembodiment, a single strand break is formed in the strand of the targetnucleic acid other than the strand to which the targeting domain of saidgRNA is complementary.

In an embodiment, the eaCas9 molecule catalyzes a double strand break.

In some embodiments, the eaCas9 molecule comprises HNH-like domaincleavage activity but has no, or no significant, N-terminal RuvC-likedomain cleavage activity. In other embodiments, the said eaCas9 moleculeis an HNH-like domain nickase, e.g., the eaCas9 molecule comprises amutation at D10, e.g., D10A. In other embodiments, the eaCas9 moleculecomprises N-terminal RuvC-like domain cleavage activity but has no, orno significant, HNH-like domain cleavage activity. In anotherembodiment, the eaCas9 molecule is an N-terminal RuvC-like domainnickase, e.g., the eaCas9 molecule comprises a mutation at H840, e.g.,H840A. In another embodiment, the eaCas9 molecule is an N-terminalRuvC-like domain nickase, e.g., the eaCas9 molecule comprises a mutationat H863, e.g., H863A.

A nucleic acid disclosed herein may comprise (a) a sequence that encodesa gRNA molecule comprising a targeting domain that is complementary witha target domain in the CEP290 gene as disclosed herein; and (b) asequence that encodes a Cas9 molecule.

A nucleic acid disclosed herein may comprise (a) a sequence that encodesa gRNA molecule comprising a targeting domain that is complementary witha target domain in the CEP290 gene as disclosed herein; (b) a sequencethat encodes a Cas9 molecule; and further comprises (c)(i) a sequencethat encodes a second gRNA molecule described herein having a targetingdomain that is complementary to a second target domain of the CEP290gene, and optionally, (ii) a sequence that encodes a third gRNA moleculedescribed herein having a targeting domain that is complementary to athird target domain of the CEP290 gene; and optionally, (iii) a sequencethat encodes a fourth gRNA molecule described herein having a targetingdomain that is complementary to a fourth target domain of the CEP290gene.

In an embodiment, a nucleic acid encodes a second gRNA moleculecomprising a targeting domain configured to provide a cleavage event,e.g., a double strand break or a single strand break, sufficiently closeto a LCA10 target position in the CEP290 gene to allow alteration, e.g.,alteration associated with NHEJ, of the LCA10 target position, eitheralone or in combination with the break positioned by said first gRNAmolecule.

In an embodiment, a nucleic acid encodes a third gRNA moleculecomprising a targeting domain configured to provide a cleavage event,e.g., a double strand break or a single strand break, sufficiently closeto a LCA10 target position in the CEP290 gene to allow alteration, e.g.,alteration associated with NHEJ, either alone or in combination with thebreak positioned by the first and/or second gRNA molecule.

In an embodiment, a nucleic acid encodes a fourth gRNA moleculecomprising a targeting domain configured to provide a cleavage event,e.g., a double strand break or a single strand break, sufficiently closeto a LCA10 target position in the CEP290 gene to allow alteration, e.g.,alteration associated with NHEJ, either alone or in combination with thebreak positioned by the first gRNA molecule, the second gRNA moleculeand/or the third gRNA molecule.

In an embodiment, a nucleic acid encodes a second gRNA moleculecomprising a targeting domain configured to provide a cleavage event,e.g., a double strand break or a single strand break, in combinationwith the break position by said first gRNA molecule, sufficiently closeto a LCA10 target position in the CEP290 gene to allow alteration, e.g.,alteration associated with NHEJ, of the a LCA10 target position in theCEP290 gene, either alone or in combination with the break positioned bysaid first gRNA molecule.

In an embodiment, a nucleic acid encodes a third gRNA moleculecomprising a targeting domain configured to provide a cleavage event,e.g., a double strand break or a single strand break, in combinationwith the break position by said first and/or second gRNA moleculesufficiently close to a LCA10 target position in the CEP290 gene toallow alteration, e.g., alteration associated with NHEJ, either alone orin combination with the break positioned by the first and/or second gRNAmolecule.

In an embodiment, a nucleic acid encodes a fourth gRNA moleculecomprising a targeting domain configured to provide a cleavage event,e.g., a double strand break or a single strand break, in combinationwith the break positioned by the first gRNA molecule, the second gRNAmolecule and/or the third gRNA molecule, sufficiently close to a LCA10target position in the CEP290 gene to allow alteration, e.g., alterationassociated with NHEJ, either alone or in combination with the breakpositioned by the first gRNA molecule, the second gRNA molecule and/orthe third gRNA molecule.

In an embodiment, the nucleic acid encodes a second gRNA molecule. Thesecond gRNA is selected to target the LCA10 target position. Optionally,the nucleic acid may encode a third gRNA, and further optionally, thenucleic acid may encode a fourth gRNA molecule.

In an embodiment, the nucleic acid encodes a second gRNA moleculecomprising a targeting domain comprising a sequence that is the same as,or differs by no more than 1, 2, 3, 4, or 5 nucleotides from, atargeting domain sequence from one of Tables 2A-2D, Tables 3A-3C, Tables4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables9A-9E, Tables 10A-10B, or Table 11. In an embodiment, the nucleic acidencodes a second gRNA molecule comprising a targeting domain selectedfrom those in Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D,Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B,or Table 11. In an embodiment, when a third or fourth gRNA molecule arepresent, the third and fourth gRNA molecules may independently comprisea targeting domain comprising a sequence that is the same as, or differsby no more than 1, 2, 3, 4, or 5 nucleotides from, a targeting domainsequence from one of Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables10A-10B, or Table 11. In a further embodiment, when a third or fourthgRNA molecule are present, the third and fourth gRNA molecules mayindependently comprise a targeting domain selected from those in Tables2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11.

In an embodiment, the nucleic acid encodes a second gRNA which is amodular gRNA, e.g., wherein one or more nucleic acid molecules encode amodular gRNA. In other embodiments, the nucleic acid encoding a secondgRNA is a chimeric gRNA. In other embodiments, when a nucleic acidencodes a third or fourth gRNA, the third and fourth gRNA may be amodular gRNA or a chimeric gRNA. When multiple gRNAs are used, anycombination of modular or chimeric gRNAs may be used.

A nucleic acid may encode a second, a third, and/or a fourth gRNA, eachindependently, comprising a targeting domain comprising 16 nucleotidesor more in length. In an embodiment, the nucleic acid encodes a secondgRNA comprising a targeting domain that is 16 nucleotides in length. Inother embodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 17 nucleotides in length. In still otherembodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 18 nucleotides in length. In still otherembodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 19 nucleotides in length. In still otherembodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 20 nucleotides in length. In still otherembodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 21 nucleotides in length. In still otherembodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 22 nucleotides in length. In still otherembodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 23 nucleotides in length. In still otherembodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 24 nucleotides in length. In still otherembodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 25 nucleotides in length. In still otherembodiments, the nucleic acid encodes a second gRNA comprising atargeting domain that is 26 nucleotides in length.

In an embodiment, the targeting domain comprises 16 nucleotides.

In an embodiment, the targeting domain comprises 17 nucleotides.

In an embodiment, the targeting domain comprises 18 nucleotides.

In an embodiment, the targeting domain comprises 19 nucleotides.

In an embodiment, the targeting domain comprises 20 nucleotides.

In an embodiment, the targeting domain comprises 21 nucleotides.

In an embodiment, the targeting domain comprises 22 nucleotides.

In an embodiment, the targeting domain comprises 23 nucleotides.

In an embodiment, the targeting domain comprises 24 nucleotides.

In an embodiment, the targeting domain comprises 25 nucleotides.

In an embodiment, the targeting domain comprises 26 nucleotides.

In an embodiment, a nucleic acid encodes a second, a third, and/or afourth gRNA, each independently, comprising from 5′ to 3′: a targetingdomain (comprising a “core domain”, and optionally a “secondarydomain”); a first complementarity domain; a linking domain; a secondcomplementarity domain; a proximal domain; and a tail domain. In someembodiments, the proximal domain and tail domain are taken together as asingle domain.

In an embodiment, a nucleic acid encodes a second, a third, and/or afourth gRNA, each independently, comprising a linking domain of no morethan 25 nucleotides in length; a proximal and tail domain, that takentogether, are at least 20 nucleotides in length; and a targeting domainof equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

In an embodiment, a nucleic acid encodes a second, a third, and/or afourth gRNA, each independently, comprising a linking domain of no morethan 25 nucleotides in length; a proximal and tail domain, that takentogether, are at least 30 nucleotides in length; and a targeting domainof equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

In an embodiment, a nucleic acid encodes a second, a third, and/or afourth gRNA, each independently, comprising a linking domain of no morethan 25 nucleotides in length; a proximal and tail domain, that takentogether, are at least 30 nucleotides in length; and a targeting domainof equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

In an embodiment, a nucleic acid encodes a second, a third, and/or afourth gRNA, each independently, comprising a linking domain of no morethan 25 nucleotides in length; a proximal and tail domain, that takentogether, are at least 40 nucleotides in length; and a targeting domainof equal to or greater than 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

In some embodiments, when the CEP290 gene is altered, e.g., by NHEJ, thenucleic acid encodes (a) a sequence that encodes a gRNA moleculecomprising a targeting domain that is complementary with a target domainin the CEP290 gene as disclosed herein; (b) a sequence that encodes aCas9 molecule; optionally, (c)(i) a sequence that encodes a second gRNAmolecule described herein having a targeting domain that iscomplementary to a second target domain of the CEP290 gene, and furtheroptionally, (ii) a sequence that encodes a third gRNA molecule describedherein having a targeting domain that is complementary to a third targetdomain of the CEP290 gene; and still further optionally, (iii) asequence that encodes a fourth gRNA molecule described herein having atargeting domain that is complementary to a fourth target domain of theCEP290 gene.

As described above, a nucleic acid may comprise (a) a sequence encodinga gRNA molecule comprising a targeting domain that is complementary witha target domain in the CEP290, and (b) a sequence encoding a Cas9molecule. In some embodiments, (a) and (b) are present on the samenucleic acid molecule, e.g., the same vector, e.g., the same viralvector, e.g., the same adeno-associated virus (AAV) vector. In anembodiment, the nucleic acid molecule is an AAV vector, e.g., an AAVvector described herein. Exemplary AAV vectors that may be used in anyof the described compositions and methods include an AAV1 vector, amodified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector,an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9vector, an AAV.rh10 vector, a modified AAV.rh10 vector, an AAV.rh32/33vector, a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modifiedAAV.rh43 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1 vector.

In other embodiments, (a) is present on a first nucleic acid molecule,e.g. a first vector, e.g., a first viral vector, e.g., a first AAVvector; and (b) is present on a second nucleic acid molecule, e.g., asecond vector, e.g., a second vector, e.g., a second AAV vector. Thefirst and second nucleic acid molecules may be AAV vectors, e.g., theAAV vectors described herein.

In other embodiments, the nucleic acid may further comprise (c)(i) asequence that encodes a second gRNA molecule as described herein. Insome embodiments, the nucleic acid comprises (a), (b) and (c)(i). Eachof (a) and (c)(i) may be present on the same nucleic acid molecule,e.g., the same vector, e.g., the same viral vector, e.g., the sameadeno-associated virus (AAV) vector. In an embodiment, the nucleic acidmolecule is an AAV vector, e.g., an AAV vectors described herein.

In other embodiments, (a) and (c)(i) are on different vectors. Forexample, (a) may be present on a first nucleic acid molecule, e.g. afirst vector, e.g., a first viral vector, e.g., a first AAV vector; and(c)(i) may be present on a second nucleic acid molecule, e.g., a secondvector, e.g., a second vector, e.g., a second AAV vector. In anembodiment, the first and second nucleic acid molecules are AAV vectors,e.g., the AAV vectors described herein.

In another embodiment, each of (a), (b), and (c)(i) are present on thesame nucleic acid molecule, e.g., the same vector, e.g., the same viralvector, e.g., an AAV vector. In an embodiment, the nucleic acid moleculeis an AAV vector. In an alternate embodiment, one of (a), (b), and(c)(i) is encoded on a first nucleic acid molecule, e.g., a firstvector, e.g., a first viral vector, e.g., a first AAV vector; and asecond and third of (a), (b), and (c)(i) is encoded on a second nucleicacid molecule, e.g., a second vector, e.g., a second vector, e.g., asecond AAV vector. The first and second nucleic acid molecule may be AAVvectors, e.g., the AAV vectors described herein.

In an embodiment, (a) is present on a first nucleic acid molecule, e.g.,a first vector, e.g., a first viral vector, a first AAV vector; and (b)and (c)(i) are present on a second nucleic acid molecule, e.g., a secondvector, e.g., a second vector, e.g., a second AAV vector. The first andsecond nucleic acid molecule may be AAV vectors, e.g., the AAV vectorsdescribed herein.

In other embodiments, (b) is present on a first nucleic acid molecule,e.g., a first vector, e.g., a first viral vector, e.g., a first AAVvector; and (a) and (c)(i) are present on a second nucleic acidmolecule, e.g., a second vector, e.g., a second vector, e.g., a secondAAV vector. The first and second nucleic acid molecule may be AAVvectors, e.g., the AAV vectors described herein.

In other embodiments, (c)(i) is present on a first nucleic acidmolecule, e.g., a first vector, e.g., a first viral vector, e.g., afirst AAV vector; and (b) and (a) are present on a second nucleic acidmolecule, e.g., a second vector, e.g., a second vector, e.g., a secondAAV vector. The first and second nucleic acid molecule may be AAVvectors, e.g., the AAV vectors described herein.

In another embodiment, each of (a), (b) and (c)(i) are present ondifferent nucleic acid molecules, e.g., different vectors, e.g.,different viral vectors, e.g., different AAV vector. For example, (a)may be on a first nucleic acid molecule, (b) on a second nucleic acidmolecule, and (c)(i) on a third nucleic acid molecule. The first, secondand third nucleic acid molecule may be AAV vectors, e.g., the AAVvectors described herein.

In another embodiment, when a third and/or fourth gRNA molecule arepresent, each of (a), (b), (c)(i), (c) (ii) and (c)(iii) may be presenton the same nucleic acid molecule, e.g., the same vector, e.g., the sameviral vector, e.g., an AAV vector. In an embodiment, the nucleic acidmolecule is an AAV vector, e.g., an AAV vector. In an alternateembodiment, each of (a), (b), (c)(i), (c)(ii) and (c)(iii) may bepresent on the different nucleic acid molecules, e.g., differentvectors, e.g., the different viral vectors, e.g., different AAV vectors.In further embodiments, each of (a), (b), (c)(i), (c) (ii) and (c)(iii)may be present on more than one nucleic acid molecule, but fewer thanfive nucleic acid molecules, e.g., AAV vectors, e.g., the AAV vectorsdescribed herein.

The nucleic acids described herein may comprise a promoter operablylinked to the sequence that encodes the gRNA molecule of (a), e.g., apromoter described herein, e.g., a promoter described in Table 20. Thenucleic acid may further comprise a second promoter operably linked tothe sequence that encodes the second, third and/or fourth gRNA moleculeof (c), e.g., a promoter described herein. The promoter and secondpromoter differ from one another. In some embodiments, the promoter andsecond promoter are the same.

The nucleic acids described herein may further comprise a promoteroperably linked to the sequence that encodes the Cas9 molecule of (b),e.g., a promoter described herein, e.g., a promoter described in Table20.

In another aspect, disclosed herein is a composition comprising (a) agRNA molecule comprising a targeting domain that is complementary with atarget domain in the CEP290 gene, as described herein. The compositionof (a) may further comprise (b) a Cas9 molecule, e.g., a Cas9 moleculeas described herein. A composition of (a) and (b) may further comprise(c) a second, third and/or fourth gRNA molecule, e.g., a second, thirdand/or fourth gRNA molecule described herein.

In another aspect, methods and compositions discussed herein, providefor treating or delaying the onset or progression of LCA10 by alteringthe LCA10 target position in the CEP290 gene.

In another aspect, disclosed herein is a method of altering a cell,e.g., altering the structure, e.g., altering the sequence, of a targetnucleic acid of a cell, comprising contacting said cell with: (a) a gRNAthat targets the CEP290 gene, e.g., a gRNA as described herein; (b) aCas9 molecule, e.g., a Cas9 molecule as described herein; andoptionally, (c) a second, third and/or fourth gRNA that targets CEP290gene, e.g., a gRNA as described herein.

In some embodiments, the method comprises contacting said cell with (a)and (b).

In some embodiments, the method comprises contacting said cell with (a),(b), and (c).

The gRNA of (a) may be selected from any of Tables 2A-2D, Tables 3A-3C,Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D,Tables 9A-9E, Tables 10A-10B, or Table 11, or a gRNA that differs by nomore than 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequencefrom any of Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D,Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B,or Table 11. The gRNA of (c) may be selected from any of Tables 2A-2D,Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D,Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11, or a gRNA thatdiffers by no more than 1, 2, 3, 4, or 5 nucleotides from, a targetingdomain sequence from any of Tables 2A-2D, Tables 3A-3C, Tables 4A-4D,Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E,Tables 10A-10B, or Table 11.

In some embodiments, the method comprises contacting a cell from asubject suffering from or likely to develop LCA10. The cell may be froma subject having a mutation at a LCA10 target position.

In some embodiments, the cell being contacted in the disclosed method isa photoreceptor cell. The contacting may be performed ex vivo and thecontacted cell may be returned to the subject's body after thecontacting step. In other embodiments, the contacting step may beperformed in vivo.

In some embodiments, the method of altering a cell as described hereincomprises acquiring knowledge of the presence of a LCA10 target positionin said cell, prior to the contacting step. Acquiring knowledge of thepresence of a LCA10 target position in the cell may be by sequencing theCEP290 gene, or a portion of the CEP290 gene.

In some embodiments, the contacting step of the method comprisescontacting the cell with a nucleic acid, e.g., a vector, e.g., an AAVvector, e.g., an AAV vector described herein, that expresses at leastone of (a), (b), and (c). In some embodiments, the contacting step ofthe method comprises contacting the cell with a nucleic acid, e.g., avector, e.g., an AAV vector, that expresses each of (a), (b), and (c).In another embodiment, the contacting step of the method comprisesdelivering to the cell a Cas9 molecule of (b) and a nucleic acid whichencodes a gRNA (a) and optionally, a second gRNA (c)(i) (and furtheroptionally, a third gRNA (c)(iv) and/or fourth gRNA (c)(iii)).

In an embodiment, contacting comprises contacting the cell with anucleic acid, e.g., a vector, e.g., an AAV vector, e.g., an AAV1 vector,a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3vector, a modified AAV3 vector, an AAV4 vector, a modified AAV4 vector,an AAV5 vector, a modified AAV5 vector, an AAV6 vector, a modified AAV6vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV9vector, an AAV.rh10 vector, a modified AAV.rh10 vector, an AAV.rh32/33vector, a modified AAV.rh32/33 vector, an AAV.rh43 vector, a modifiedAAV.rh43 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1 vector,e.g., an AAV vector described herein.

In an embodiment, contacting comprises delivering to said cell said Cas9molecule of (b), as a protein or an mRNA, and a nucleic acid whichencodes and (a) and optionally (c).

In an embodiment, contacting comprises delivering to said cell said Cas9molecule of (b), as a protein or an mRNA, said gRNA of (a), as an RNA,and optionally said second gRNA of (c), as an RNA.

In an embodiment, contacting comprises delivering to said cell said gRNAof (a) as an RNA, optionally said second gRNA of (c) as an RNA, and anucleic acid that encodes the Cas9 molecule of (b).

In another aspect, disclosed herein is a method of treating, orpreventing a subject suffering from developing, LCA10, e.g., by alteringthe structure, e.g., sequence, of a target nucleic acid of the subject,comprising contacting the subject (or a cell from the subject) with:

(a) a gRNA that targets the CEP290 gene, e.g., a gRNA disclosed herein;

(b) a Cas9 molecule, e.g., a Cas9 molecule disclosed herein; and

optionally, (c)(i) a second gRNA that targets the CEP290 gene, e.g., asecond gRNA disclosed herein, and

further optionally, (c)(ii) a third gRNA, and still further optionally,(c)(iii) a fourth gRNA that target the CEP290, e.g., a third and fourthgRNA disclosed herein.

In some embodiments, contacting comprises contacting with (a) and (b).

In some embodiments, contacting comprises contacting with (a), (b), and(c)(i).

In some embodiments, contacting comprises contacting with (a), (b),(c)(i) and (c)(ii).

In some embodiments, contacting comprises contacting with (a), (b),(c)(i), (c)(ii) and (c)(iii).

The gRNA of (a) or (c) (e.g., (c)(i), (c)(ii), or (c)(iii)) may beindependently selected from any of Tables 2A-2D, Tables 3A-3C, Tables4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables9A-9E, Tables 10A-10B, or Table 11, or a gRNA that differs by no morethan 1, 2, 3, 4, or 5 nucleotides from, a targeting domain sequence fromany of Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, orTable 11.

In an embodiment, said subject is suffering from, or likely to developLCA10. In an embodiment, said subject has a mutation at a LCA10 targetposition.

In an embodiment, the method comprises acquiring knowledge of thepresence of a mutation at a LCA10 target position in said subject.

In an embodiment, the method comprises acquiring knowledge of thepresence of a mutation a LCA10 target position in said subject bysequencing the CEP290 gene or a portion of the CEP290 gene.

In an embodiment, the method comprises altering the LCA10 targetposition in the CEP290 gene.

In an embodiment, a cell of said subject is contacted ex vivo with (a),(b) and optionally (c). In an embodiment, said cell is returned to thesubject's body.

In an embodiment, the method comprises introducing a cell into saidsubject's body, wherein said cell subject was contacted ex vivo with(a), (b) and optionally (c).

In an embodiment, the method comprises said contacting is performed invivo. In an embodiment, the method comprises sub-retinal delivery. In anembodiment, contacting comprises sub-retinal injection. In anembodiment, contacting comprises intra-vitreal injection.

In an embodiment, contacting comprises contacting the subject with anucleic acid, e.g., a vector, e.g., an AAV vector described herein,e.g., a nucleic acid that encodes at least one of (a), (b), andoptionally (c).

In an embodiment, contacting comprises delivering to said subject saidCas9 molecule of (b), as a protein or mRNA, and a nucleic acid whichencodes and (a) and optionally (c).

In an embodiment, contacting comprises delivering to said subject saidCas9 molecule of (b), as a protein or mRNA, said gRNA of (a), as an RNA,and optionally said second gRNA of (c), as an RNA.

In an embodiment, contacting comprises delivering to said subject saidgRNA of (a), as an RNA, optionally said second gRNA of (c), as an RNA,and a nucleic acid that encodes the Cas9 molecule of (b).

In another aspect, disclosed herein is a reaction mixture comprising agRNA, a nucleic acid, or a composition described herein, and a cell,e.g., a cell from a subject having, or likely to develop LCA10, or asubject having a mutation at a LCA10 target position.

In another aspect, disclosed herein is a kit comprising, (a) a gRNAmolecule described herein, or a nucleic acid that encodes said gRNA, andone or more of the following:

(b) a Cas9 molecule, e.g., a Cas9 molecule described herein, or anucleic acid or mRNA that encodes the Cas9;

(c)(i) a second gRNA molecule, e.g., a second gRNA molecule describedherein or a nucleic acid that encodes (c)(i);

(c)(ii) a third gRNA molecule, e.g., a second gRNA molecule describedherein or a nucleic acid that encodes (c)(ii); or

(c)(iii) a fourth gRNA molecule, e.g., a second gRNA molecule describedherein or a nucleic acid that encodes (c)(iii).

In an embodiment, the kit comprises nucleic acid, e.g., an AAV vector,e.g., an AAV vector described herein, that encodes one or more of (a),(b), (c)(i), (c)(ii), and (c)(iii). In an embodiment, the kit furthercomprises a governing gRNA molecule, or a nucleic acid that encodes agoverning gRNA molecule.

In yet another aspect, disclosed herein is a gRNA molecule, e.g., a gRNAmolecule described herein, for use in treating LCA10 in a subject, e.g.,in accordance with a method of treating LCA10 as described herein.

In an embodiment, the gRNA molecule in used in combination with a Cas9molecule, e.g., a Cas9 molecule described herein. Additionally oralternatively, in an embodiment, the gRNA molecule is used incombination with a second, third and/or fourth gRNA molecule, e.g., asecond, third and/or fourth gRNA molecule described herein.

In still another aspect, disclosed herein is use of a gRNA molecule,e.g., a gRNA molecule described herein, in the manufacture of amedicament for treating LCA10 in a subject, e.g., in accordance with amethod of treating LCA10 as described herein.

In an embodiment, the medicament comprises a Cas9 molecule, e.g., a Cas9molecule described herein. Additionally or alternatively, in anembodiment, the medicament comprises a second, third and/or fourth gRNAmolecule, e.g., a second, third and/or fourth gRNA molecule describedherein.

In one aspect, disclosed herein is a recombinant adenovirus-associatedvirus (AAV) genome comprising the following components:

wherein the left ITR component comprises, or consists of, a nucleotidesequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotides from, or has at least 90%, 92%, 94%, 96%,98%, or 99% homology with, any of the left ITR nucleotide sequencesdisclosed in Table 25, or any of the nucleotide sequences of SEQ ID NOs:407-415;

wherein the spacer 1 component comprises, or consists of, a nucleotidesequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 416;

wherein the PIII promoter component comprises, or consists of, an RNApolymerase III promoter sequence;

wherein the gRNA component comprises a targeting domain and a scaffolddomain,

-   -   wherein the targeting domain is 16-26 nucleotides in length, and        comprises, or consists of, a targeting domain sequence disclosed        herein, e.g., in any of Tables 2A-2D, Tables 3A-3C, Tables        4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D,        Tables 9A-9E, Tables 10A-10B, or Table 11; and    -   wherein the scaffold domain (also referred to as a tracr domain        in FIGS. 20A-25F) comprises, or consists of, a nucleotide        sequence that is the same as, differs by no more than 1, 2, 3,        4, or 5 nucleotides from, or has at least has at least 90%, 92%,        94%, 96%, 98%, or 99% homology with, a nucleotide sequence of        SEQ ID NO: 418;

wherein the spacer 2 component comprises, or consists of, a nucleotidesequence having 0 to 150 nucleotides in length e.g., SEQ ID NO: 419;

wherein the PII promoter component comprises, or consists of, apolymerase II promoter sequence, e.g., a constitutive or tissue specificpromoter, e.g., a promoter disclosed in Table 20;

wherein the N-ter NLS component comprises, or consists of, a nucleotidesequence that is the same as, differs by no more than 1, 2, 3, 4, or 5nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%,or 99% homology with, the nucleotide sequence of SEQ ID NO: 420 or anucleotide sequence that encodes the amino acid sequence of SEQ ID NO:434;

wherein the Cas9 component comprises, or consists of, a nucleotidesequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotides from, or has at least 90%, 92%, 94%, 96%,98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 421 ora nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:26;

wherein the C-ter NLS component comprises, or consists of, a nucleotidesequence that is the same as, differs by no more than 1, 2, 3, 4, or 5nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%,or 99% homology with, the nucleotide sequence of SEQ ID NO: 422 or anucleotide sequence that encodes the amino acid sequence of SEQ ID NO:434;

wherein the poly(A) signal component comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, any of thenucleotide sequences disclosed in Table 27, or any of the nucleotidesequences of SEQ ID NOs: 424, 455 or 456;

wherein the spacer 3 component comprises, or consists of, a nucleotidesequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 425;and

wherein the right ITR component comprises, or consists of, a nucleotidesequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotides from, or has at least has at least 90%, 92%,94%, 96%, 98%, or 99% homology with, any of the right ITR nucleotidesequences disclosed in Table 25, or any of the nucleotide sequences ofSEQ ID NOs: 436-444.

In an embodiment, the left ITR component comprises, or consists of, anucleotide sequence that is the same as any of the nucleotide sequencesof SEQ ID NOs: 407-415.

In an embodiment, the spacer 1 component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 416.

In an embodiment, the PIII promoter component is a U6 promotercomponent.

In an embodiment, the U6 promoter component comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 417;

In an embodiment, the U6 promoter component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 417.

In an embodiment, the PIII promoter component is an H1 promotercomponent that comprises an H1 promoter sequence.

In an embodiment, the PIII promoter component is a tRNA promotercomponent that comprises a tRNA promoter sequence.

In an embodiment, the targeting domain comprises, or consists of, anucleotide sequence that is the same as a nucleotide sequence selectedfrom Table 11.

In an embodiment, the gRNA scaffold domain comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 418.

In an embodiment, the spacer 2 component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 419;

In an embodiment, the PII promoter component is a CMV promotercomponent, and comprises, or consists of, a nucleotide sequence that isthe same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%,or 99% homology with, the nucleotide sequence of SEQ ID NO: 401. In anembodiment, the PII promoter comprises, or consists of, a nucleotidesequence that is the same as the nucleotide sequence of SEQ ID NO: 401.

In an embodiment, the PII promoter component is an EFS promotercomponent, and comprises, or consists of, a nucleotide sequence that isthe same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%,or 99% homology with, the nucleotide sequence of SEQ ID NO: 402. In anembodiment, the PII promoter comprises, or consists of, a nucleotidesequence that is the same as the nucleotide sequence of SEQ ID NO: 402.

In an embodiment, the PII promoter component is a GRK1 promoter (e.g., ahuman GRK1 promoter) component, and comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 403. In an embodiment, the PII promotercomprises, or consists of, a nucleotide sequence that is the same as thenucleotide sequence of SEQ ID NO: 403.

In an embodiment, the PII promoter component is a CRX promoter (e.g., ahuman CRX promoter) component, and comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 404. In an embodiment, the PII promotercomprises, or consists of, a nucleotide sequence that is the same as thenucleotide sequence of SEQ ID NO: 404.

In an embodiment, the PII promoter component is an NRL promoter (e.g., ahuman NRL promoter) component, and comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 405. In an embodiment, the PII promotercomprises, or consists of, a nucleotide sequence that is the same as thenucleotide sequence of SEQ ID NO: 405.

In an embodiment, the PII promoter component is an RCVRN promoter (e.g.,a human RCVRN promoter) component, and comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 406. In an embodiment, the PII promotercomprises, or consists of, a nucleotide sequence that is the same as thenucleotide sequence of SEQ ID NO: 406.

In an embodiment, the N-ter NLS component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 420 or a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 434.

In an embodiment, the Cas9 component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 421 or a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 26.

In an embodiment, the C-ter NLS component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 422 or a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 434.

In an embodiment, the poly(A) signal component comprises, or consistsof, a nucleotide sequence that is the same as any of the nucleotidesequences disclosed in Table 27, or any of the nucleotide sequences ofSEQ ID NOs: 424, 455 or 456. In an embodiment, the poly(A) signalcomponent comprises, or consists of, a nucleotide sequence that is thesame as the nucleotide sequence of SEQ ID NO: 424.

In an embodiment, the spacer 3 component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 425.

In an embodiment, the right ITR component comprises, or consists of, anucleotide sequence that is the same as any of the nucleotide sequencesof SEQ ID NOs: 436-444.

In an embodiment, the recombinant AAV genome further comprises a secondgRNA component comprising a targeting domain and a scaffold domain,

wherein the targeting domain consists of a targeting domain sequencedisclosed herein, in any of Tables 2A-2D, Tables 3A-3C, Tables 4A-4D,Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E,Tables 10A-10B, or Table 11; and

wherein the scaffold domain (also referred to as a tracr domain in FIGS.20A-25F) comprises, or consists of, a nucleotide sequence that is thesame as, or differs by no more than 1, 2, 3, 4, or 5 nucleotides from,or has at least has at least 90%, 92%, 94%, 96%, 98%, or 99% homologywith, the nucleotide sequence of SEQ ID NO: 418.

In an embodiment, the targeting domain of the second gRNA componentcomprises, or consists of, a nucleotide sequence that is the same as anucleotide sequence selected from Table 11. In an embodiment, the secondgRNA component is between the first gRNA component and the spacer 2component.

In an embodiment, the second gRNA component has the same nucleotidesequence as the first gRNA component. In another embodiment, the secondgRNA component has a nucleotide sequence that is different from thesecond gRNA component.

In an embodiment, the recombinant AAV genome further comprises a secondPIII promoter component that comprises, or consists of, an RNApolymerase III promoter sequence;

In an embodiment, the recombinant AAV genome further comprises a secondPIII promoter component (e.g., a second U6 promoter component) betweenthe first gRNA component and the second gRNA component.

In an embodiment, the second PIII promoter component (e.g., the secondU6 promoter component) has the same nucleotide sequence as the firstPIII promoter component (e.g., the first U6 promoter component). Inanother embodiment, the second PIII promoter component (e.g., the secondU6 promoter component) has a nucleotide sequence that is different fromthe first PIII promoter component (e.g. the first U6 promotercomponent).

In an embodiment, the PIII promoter component is an H1 promotercomponent that comprises an H1 promoter sequence.

In an embodiment, the PIII promoter component is a tRNA promotercomponent that comprises a tRNA promoter sequence.

In an embodiment, the recombinant AAV genome further comprises a spacer4 component between the first gRNA component and the second PIIIpromoter component (e.g., the second U6 promoter component). In anembodiment, the spacer 4 component comprises, or consists of, anucleotide sequence having 0 to 150 nucleotides in length, e.g., SEQ IDNO: 427. In an embodiment, the spacer 4 component comprises, or consistsof, a nucleotide sequence that is the same as the nucleotide sequence ofSEQ ID NO: 427.

In an embodiment, the recombinant AAV genome comprises the followingcomponents:

In an embodiment, the recombinant AAV genome further comprises anaffinity tag component (e.g., 3×FLAG component), wherein the affinitytag component (e.g., 3×FLAG component) comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, or 5 nucleotides from, or has at least has at least 90%, 92%, 94%,96%, 98%, or 99% homology with, the nucleotides sequence of SEQ ID NO:423, or a nucleotide sequence encoding any of the amino acid sequencesdisclosed in Table 26 or any of the amino acid sequences of SEQ ID NOs:426 or 451-454.

In an embodiment, the affinity tag component (e.g., 3×FLAG component) isbetween the C-ter NLS component and the poly(A) signal component. In anembodiment, the an affinity tag component (e.g., 3×FLAG component)comprises, or consists of, a nucleotide sequence that is the same as,the nucleotides sequence of SEQ ID NO: 423, or a nucleotide sequenceencoding any of the amino acid sequences of SEQ ID NOs: 426 or 451-454.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 401, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 402, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 403, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 404, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 405, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 406, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome further comprises SEQ IDNOs: 416, 419, and 425, and, optionally, SEQ ID NO 427.

In an embodiment, the recombinant AAV genome further comprises thenucleotide sequence of SEQ ID NO: 423.

In an embodiment, the recombinant AAV genome comprises or consists ofone or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all) of thecomponent sequences shown in FIG. 19A-19G, 20A-20F, 21A-21F, 22A-22F,23A-23F, or 24A-24F, Tables 20 or 25-27, or any of the nucleotidesequences of SEQ ID NOs: 428-433 or 436-444.

In another aspect, disclosed herein is a recombinantadenovirus-associated virus (AAV) genome comprising the followingcomponents:

wherein the left ITR component comprises, or consists of, a nucleotidesequence that is the same as, or differs by no more than 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 nucleotides from, or has at least 90%, 92%, 94%, 96%,98%, or 99% homology with, any of the left ITR nucleotide sequencesdisclosed in Table 25, or any of the nucleotide sequences of SEQ ID NOs:407-415;

wherein the spacer 1 component comprises, or consists of, a nucleotidesequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 416;

wherein the first PIII promoter component (e.g., a first U6 promotercomponent) comprises, or consists of, a nucleotide sequence that is thesame as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%,or 99% homology with, the nucleotide sequence of SEQ ID NO: 417;

wherein the first gRNA component comprises a targeting domain and ascaffold domain,

-   -   wherein the targeting domain is 16-26 nucleotides in length, and        comprises, or consists of, a targeting domain sequence disclosed        herein, e.g., in any of Tables 2A-2D, Tables 3A-3C, Tables        4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D,        Tables 9A-9E, Tables 10A-10B, or Table 11; and    -   wherein the scaffold domain (also referred to herein as a tracr        domain in FIGS. 19A-24F) comprises, or consists of, a nucleotide        sequence that is the same as, or differs by no more than 1, 2,        3, 4, or 5 nucleotides from, or has at least has at least 90%,        92%, 94%, 96%, 98%, or 99% homology with, the nucleotide        sequence of SEQ ID NO: 418;

wherein the spacer 4 component comprises, or consists of, a nucleotidesequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 427.

wherein the second gRNA component comprises a targeting domain and ascaffold domain,

-   -   wherein the targeting domain of the second gRNA component is        16-26 nucleotides in length and comprises, or consists of, a        targeting domain sequence disclosed herein, e.g., in any of        Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables        6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B,        or Table 11; and    -   wherein the scaffold domain (also referred to as a tracr domain        in FIGS. 19A-24F) of the second gRNA component comprises, or        consists of, a nucleotide sequence that is the same as, or        differs by no more than 1, 2, 3, 4, or 5 nucleotides from, or        has at least has at least 90%, 92%, 94%, 96%, 98%, or 99%        homology with, the nucleotide sequence of SEQ ID NO: 418.

wherein the spacer 2 component comprises, or consists of, a nucleotidesequence having 0 to 150 nucleotides in length e.g., SEQ ID NO: 419;

wherein the PII promoter component comprises, or consists of, apolymerase II promoter sequence, e.g., a constitutive or tissue specificpromoter, e.g., a promoter disclosed in Table 20;

wherein the N-ter NLS component comprises, or consists of, a nucleotidesequence that is the same as, differs by no more than 1, 2, 3, 4, or 5nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%,or 99% homology with, the nucleotide sequence of SEQ ID NO: 420 or anucleotide sequence that encodes the amino acid sequence of SEQ ID NO:434;

wherein the Cas9 component comprises, or consists of, a nucleotidesequence that is the same as, differs by no more than 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotides from, or has at least 90%, 92%, 94%, 96%,98%, or 99% homology with, the nucleotide sequence of SEQ ID NO: 421 ora nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:26;

wherein the C-ter NLS component comprises, or consists of, a nucleotidesequence that is the same as, differs by no more than 1, 2, 3, 4, or 5nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%,or 99% homology with, the nucleotide sequence of SEQ ID NO: 422 or anucleotide sequence that encodes the amino acid sequence of SEQ ID NO:434;

wherein the poly(A) signal component comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, any of thenucleotide sequences disclosed in Table 27, or any of the nucleotidesequence of SEQ ID NO: 424, 455 or 456;

wherein the spacer 3 component comprises, or consists of, a nucleotidesequence having 0 to 150 nucleotides in length, e.g., SEQ ID NO: 425;and

wherein the right ITR component comprises, or consists of, a nucleotidesequence that is the same as, or differs by no more than 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 nucleotides from, or has at least has at least 90%,92%, 94%, 96%, 98%, or 99% homology with, any of the right ITRnucleotide sequences disclosed in Table 25, or SEQ ID NOs: 436-444.

In an embodiment, the left ITR component comprises, or consists of, anucleotide sequence that is the same as any of the nucleotide sequencesof SEQ ID NOs: 407-415.

In an embodiment, the spacer 1 component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 416.

In an embodiment, the first PIII promoter component (e.g., the first U6promoter component) comprises, or consists of, a nucleotide sequencethat is the same as the nucleotide sequence of SEQ ID NO: 417.

In an embodiment, the first PIII promoter is an H1 promoter componentthat comprises an H1 promoter sequence. In another embodiment, the firstPIII promoter is a tRNA promoter component that comprises a tRNApromoter sequence.

In an embodiment, the targeting domain of the first gRNA componentcomprises, or consists of, a nucleotide sequence that is the same as anucleotide sequence selected from Table 11.

In an embodiment, the gRNA scaffold domain of the first gRNA componentcomprises, or consists of, a nucleotide sequence that is the same as thenucleotide sequence of SEQ ID NO: 418.

In an embodiment, the spacer 4 component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 427.

In an embodiment, the second PIII promoter component (e.g., the first U6promoter component) has the same nucleotide sequence as the first PIIIpromoter component (e.g., the first U6 promoter component). In anotherembodiment, the second PIII promoter component (e.g., the second U6promoter component) has a nucleotide sequence that is different from thefirst PIII promoter component (e.g., the first U6 promoter component).

In an embodiment, the second PIII promoter is an H1 promoter componentthat comprises an H1 promoter sequence. In another embodiment, thesecond PIII promoter is a tRNA promoter component that comprises a tRNApromoter sequence.

In an embodiment, the targeting domain of the second gRNA componentcomprises, or consists of, a nucleotide sequence that is the same as anucleotide sequence selected from Table 11.

In an embodiment, the second gRNA component has the same nucleotidesequence as the first gRNA component. In another embodiment, the secondgRNA component has a nucleotide sequence that is different from thesecond gRNA component.

In an embodiment, the spacer 2 component comprises, or consists of, anucleotide sequence having 0 to 150 nucleotides in length e.g., SEQ IDNO: 419;

In an embodiment, the PII promoter component is a CMV promotercomponent, and comprises, or consists of, a nucleotide sequence that isthe same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%,or 99% homology with, the nucleotide sequence of SEQ ID NO: 401. In anembodiment, the PII promoter comprises, or consists of, a nucleotidesequence that is the same as the nucleotide sequence of SEQ ID NO: 401.

In an embodiment, the PII promoter component is an EFS promotercomponent, and comprises, or consists of, a nucleotide sequence that isthe same as, differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotides from, or has at least has at least 90%, 92%, 94%, 96%, 98%,or 99% homology with, the nucleotide sequence of SEQ ID NO: 402. In anembodiment, the PII promoter comprises, or consists of, a nucleotidesequence that is the same as the nucleotide sequence of SEQ ID NO: 402.

In an embodiment, the PII promoter component is a GRK1 promoter (e.g., ahuman GRK1 promoter) component, and comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 403. In an embodiment, the PII promotercomprises, or consists of, a nucleotide sequence that is the same as thenucleotide sequence of SEQ ID NO: 403.

In an embodiment, the PII promoter component is a CRX promoter (e.g., ahuman CRX promoter) component, and comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 404. In an embodiment, the PII promotercomprises, or consists of, a nucleotide sequence that is the same as thenucleotide sequence of SEQ ID NO: 404.

In an embodiment, the PII promoter component is an NRL promoter (e.g., ahuman NRL promoter) component, and comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 405. In an embodiment, the PII promotercomprises, or consists of, a nucleotide sequence that is the same as thenucleotide sequence of SEQ ID NO: 405.

In an embodiment, the PII promoter component is an RCVRN promoter (e.g.,a human RCVRN promoter) component, and comprises, or consists of, anucleotide sequence that is the same as, differs by no more than 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 406. In an embodiment, the PII promotercomprises, or consists of, a nucleotide sequence that is the same as thenucleotide sequence of SEQ ID NO: 406.

In an embodiment, the N-ter NLS component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 420 or a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 434.

In an embodiment, the Cas9 component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 421 or a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 26.

In an embodiment, the C-ter NLS component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 422 or a nucleotide sequence encoding the amino acid sequence ofSEQ ID NO: 434.

In an embodiment, the poly(A) signal component comprises, or consistsof, a nucleotide sequence that is the same as any of the nucleotidesequences disclosed in Table 27, or any of the nucleotide sequences ofSEQ ID NOs: 424, 455 or 456. In an embodiment, the poly(A) signalcomponent comprises, or consists of, a nucleotide sequence that is thesame as the nucleotide sequence of SEQ ID NO: 424.

In an embodiment, the spacer 3 component comprises, or consists of, anucleotide sequence that is the same as the nucleotide sequence of SEQID NO: 425.

In an embodiment, the right ITR component comprises, or consists of, anucleotide sequence that is the same as any of the nucleotide sequencesdisclosed in Table 25, or any of the nucleotide sequences of SEQ ID NOs:436-444.

In an embodiment, the recombinant AAV genome further comprises anaffinity tag component (e.g., a 3×FLAG component). In an embodiment, theaffinity tag component (e.g., the 3×FLAG component) comprises, orconsists of, a nucleotide sequence that is the same as, differs by nomore than 1, 2, 3, 4, or 5 nucleotides from, or has at least has atleast 90%, 92%, 94%, 96%, 98%, or 99% homology with, the nucleotidesequence of SEQ ID NO: 423, or a nucleotide sequence encoding any of theamino acid sequences disclosed in Table 26 or any of the amino acidsequences of SEQ ID NO: 426 or 451-454.

In an embodiment, the affinity tag component (e.g., the 3×FLAGcomponent) is between the C-ter NLS component and the poly(A) signalcomponent. In an embodiment, the affinity tag component (e.g., the3×FLAG component) comprises, or consists of, a nucleotide sequence thatis the same as, the nucleotide sequence of SEQ ID NO: 423 or anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 426.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 401, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 402, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 403, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 404, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 405, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome comprises the nucleotidesequences of SEQ ID NOs: 408, 417, 418, 406, 420, 421, 422, 424, and437.

In an embodiment, the recombinant AAV genome further comprises thenucleotide sequences of SEQ ID NO: 416, 419, 425, and 427.

In an embodiment, the recombinant AAV genome further comprises thenucleotide sequence of SEQ ID NO: 423.

In an embodiment, the recombinant AAV genome comprises any of thenucleotide sequences of SEQ ID NOs: 428-433.

In an embodiment, the recombinant AAV genome comprises, or consists of,a nucleotide sequence that is the same as, differs by no more than 100,200, 300, 400, or 500 nucleotides from, or has at least has at least90%, 92%, 94%, 96%, 98%, or 99% homology with any of the nucleotidesequences shown in FIG. 19A-19G, 20A-20F, 21A-21F, 22A-22F, 23A-23F, or24A-24F, or any of the nucleotide sequences of SEQ ID NOs: 428-433 or436-444.

In an embodiment, the recombinant AAV genome comprises, or consists of,a nucleotide sequence that is the same as any of the nucleotidesequences shown in FIG. 19A-19G, 20A-20F, 21A-21F, 22A-22F, 23A-23F, or24A-24F, or any of the nucleotide sequences of SEQ ID NOs: 428-433 or436-444.

In an embodiment, the recombinant AAV genome comprises or consists ofone or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all) of thecomponent sequences shown in FIG. 19A-19G, 20A-20F, 21A-21F, 22A-22F,23A-23F, or 24A-24F, or Tables 20 or 25-27, or any of the nucleotidesequences of SEQ ID NOs: 428-433 or 436-444.

Unless otherwise indicated, when components of a recombinant AAV genomeare described herein, the order can be as provided, but other orders areincluded as well. In other words, in an embodiment, the order is as setout in the text, but in other embodiments, the order can be different.

It is understood that the recombinant AAV genomes disclosed herein canbe single stranded or double stranded. Disclosed herein are also thereverse, complementary form of any of the recombinant AAV genomesdisclosed herein, and the double stranded form thereof.

In another aspect, disclosed herein is a nucleic acid molecule (e.g., anexpression vector) that comprises a recombinant AAV genome disclosedherein. In an embodiment, the nucleic acid molecule further comprises anucleotide sequence that encodes an antibiotic resistant gene (e.g., anAmp resistant gene). In an embodiment, the nucleic acid molecule furthercomprises replication origin sequence (e.g., a ColE1 origin, an M13origin, or both).

In another aspect, disclosed herein is a recombinant AAV viral particlecomprising a recombinant AAV genome disclosed herein.

In an embodiment, the recombinant AAV viral particle has any of theserotype disclosed herein, e.g., in Table 25, or a combination thereof.In another embodiment, the recombinant AAV viral particle has a tissuespecificity of retinal pigment epithelium cells, photoreceptors,horizontal cells, bipolar cells, amacrine cells, ganglion cells, or acombination thereof.

In another aspect, disclosed herein is a method of producing arecombinant AAV viral particle disclosed herein comprising providing arecombinant AAV genome disclosed herein and one or more capsid proteinsunder conditions that allow for assembly of an AAV viral particle.

In another aspect, disclosed herein is a method of altering a cellcomprising contacting the cell with a recombinant AAV viral particledisclosed herein.

In another aspect, disclosed herein is a method of treating a subjecthaving or likely to develop LCA10 comprising contacting the subject (ora cell from the subject) with a recombinant viral particle disclosedherein.

In another aspect, disclosed herein is a recombinant AAV viral particlecomprising a recombinant AAV genome disclosed herein for use in treatingLCA10 in a subject.

In another aspect, disclosed herein is use of a recombinant AAV viralparticle comprising a recombinant AAV genome disclosed herein in themanufacture of a medicament for treating LCA10 in a subject.

The gRNA molecules and methods, as disclosed herein, can be used incombination with a governing gRNA molecule, comprising a targetingdomain which is complementary to a target domain on a nucleic acid thatencodes a component of the CRISPR/Cas system introduced into a cell orsubject. In an embodiment, the governing gRNA molecule targets a nucleicacid that encodes a Cas9 molecule or a nucleic acid that encodes atarget gene gRNA molecule. In an embodiment, the governing gRNAcomprises a targeting domain that is complementary to a target domain ina sequence that encodes a Cas9 component, e.g., a Cas9 molecule ortarget gene gRNA molecule. In an embodiment, the target domain isdesigned with, or has, minimal homology to other nucleic acid sequencesin the cell, e.g., to minimize off-target cleavage. For example, thetargeting domain on the governing gRNA can be selected to reduce orminimize off-target effects. In an embodiment, a target domain for agoverning gRNA can be disposed in the control or coding region of a Cas9molecule or disposed between a control region and a transcribed region.In an embodiment, a target domain for a governing gRNA can be disposedin the control or coding region of a target gene gRNA molecule ordisposed between a control region and a transcribed region for a targetgene gRNA. While not wishing to be bound by theory, in an embodiment, itis believed that altering, e.g., inactivating, a nucleic acid thatencodes a Cas9 molecule or a nucleic acid that encodes a target genegRNA molecule can be effected by cleavage of the targeted nucleic acidsequence or by binding of a Cas9 molecule/governing gRNA moleculecomplex to the targeted nucleic acid sequence.

The compositions, reaction mixtures and kits, as disclosed herein, canalso include a governing gRNA molecule, e.g., a governing gRNA moleculedisclosed herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Headings, including numeric and alphabetical headings and subheadings,are for organization and presentation and are not intended to belimiting.

Other features and advantages of the invention will be apparent from thedetailed description, drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G are representations of several exemplary gRNAs.

FIG. 1A depicts a modular gRNA molecule derived in part (or modeled on asequence in part) from Streptococcus pyogenes (S. pyogenes) as aduplexed structure (SEQ ID NOs: 42 and 43, respectively, in order ofappearance);

FIG. 1B depicts a unimolecular (or chimeric) gRNA molecule derived inpart from S. pyogenes as a duplexed structure (SEQ ID NO: 44);

FIG. 1C depicts a unimolecular gRNA molecule derived in part from S.pyogenes as a duplexed structure (SEQ ID NO: 45);

FIG. 1D depicts a unimolecular gRNA molecule derived in part from S.pyogenes as a duplexed structure (SEQ ID NO: 46);

FIG. 1E depicts a unimolecular gRNA molecule derived in part from S.pyogenes as a duplexed structure (SEQ ID NO: 47);

FIG. 1F depicts a modular gRNA molecule derived in part fromStreptococcus thermophilus (S. thermophilus) as a duplexed structure(SEQ ID NOs: 48 and 49, respectively, in order of appearance);

FIG. 1G depicts an alignment of modular gRNA molecules of S. pyogenes(SEQ ID NOs: 42 and 52) and S. thermophilus (SEQ ID NOs: 48 and 49).

FIGS. 2A-2G depict an alignment of Cas9 sequences from Chylinski 2013.The N-terminal RuvC-like domain is boxed and indicated with a “Y”. Theother two RuvC-like domains are boxed and indicated with a “B”. TheHNH-like domain is boxed and indicated by a “G”. Sm: S. mutans (SEQ IDNO: 1); Sp: S. pyogenes (SEQ ID NO: 2); St: S. thermophilus (SEQ ID NO:3); Li: L. innocua (SEQ ID NO: 4). Motif: this is a motif based on thefour sequences: residues conserved in all four sequences are indicatedby single letter amino acid abbreviation; “*” indicates any amino acidfound in the corresponding position of any of the four sequences; and“-” indicates any amino acid, e.g., any of the 20 naturally occurringamino acids (SEQ ID NO: 2804).

FIGS. 3A-3B show an alignment of the N-terminal RuvC-like domain fromthe Cas9 molecules disclosed in Chylinski 2013 (SEQ ID NOs: 54, 56, and58-103, respectively, in order of appearance). The last line of FIG. 3Bidentifies 4 highly conserved residues.

FIGS. 4A-4B show an alignment of the N-terminal RuvC-like domain fromthe Cas9 molecules disclosed in Chylinski 2013 with sequence outliersremoved. The last line of FIG. 4B identifies 3 highly conservedresidues.

FIGS. 5A-5C show an alignment of the HNH-like domain from the Cas9molecules disclosed in Chylinski 2013 (SEQ ID NOs: 178-252,respectively, in order of appearance). The last line of FIG. 5Cidentifies conserved residues.

FIGS. 6A-6B show an alignment of the HNH-like domain from the Cas9molecules disclosed in Chylinski 2013 with sequence outliers removed.The last line of FIG. 6B identifies 3 highly conserved residues.

FIGS. 7A-7B depict an alignment of Cas9 sequences from S. pyogenes andNeisseria meningitidis (N. meningitidis). The N-terminal RuvC-likedomain is boxed and indicated with a “Y”. The other two RuvC-likedomains are boxed and indicated with a “B”. The HNH-like domain is boxedand indicated with a “G”. Sp: S. pyogenes; Nm: N. meningitidis. Motif:this is a motif based on the two sequences: residues conserved in bothsequences are indicated by a single amino acid designation; “*”indicates any amino acid found in the corresponding position of any ofthe two sequences; “-” indicates any amino acid, e.g., any of the 20naturally occurring amino acids, and “-” indicates any amino acid, e.g.,any of the 20 naturally occurring amino acids, or absent.

FIG. 8 shows a nucleic acid sequence encoding Cas9 of N. meningitidis(SEQ ID NO: 303). Sequence indicated by an “R” is an SV40 NLS; sequenceindicated as “G” is an HA tag; and sequence indicated by an “0” is asynthetic NLS sequence; the remaining (unmarked) sequence is the openreading frame (ORF).

FIGS. 9A-9B are schematic representations of the domain organization ofS. pyogenes Cas 9. FIG. 9A shows the organization of the Cas9 domains,including amino acid positions, in reference to the two lobes of Cas9(recognition (REC) and nuclease (NUC) lobes). FIG. 9B shows the percenthomology of each domain across 83 Cas9 orthologs.)

FIG. 10 shows the nucleotide locations of the Alu repeats, cryptic exonand point mutation, c.2991+1655 A to G in the human CEP290 locus. “X”indicates the cryptic exon. The blue triangle indicates the LCA targetposition c.2991+1655A to G.

FIG. 11A-11B show the rates of indels induced by various gRNAs at theCEP290 locus. FIG. 11A shows gene editing (% indels) as assessed bysequencing for S. pyogenes and S. aureus gRNAs when co-expressed withCas9 in patient-derived IVS26 primary fibroblasts. FIG. 11B shows geneediting (% indels) as assessed by sequencing for S. aureus gRNAs whenco-expressed with Cas9 in HEK293 cells.

FIGS. 12A-12B show changes in expression of the wild-type and mutant(including cryptic exon) alleles of CEP290 in cells transfected withCas9 and the indicated gRNA pairs. Total RNA was isolated from modifiedcells and qRT-PCR with Taqman primer-probe sets was used to quantifyexpression. Expression is normalized to the Beta-Actin housekeeping geneand each sample is normalized to the GFP control sample (cellstransfected with only GFP). Error bars represent standard deviation of 4technical replicates.

FIG. 13 shows changes in gene expression of the wild-type and mutant(including cryptic exon) alleles of CEP290 in cells transfected withCas9 and pairs of gRNAs shown to have in initial qRT-PCR screening.Total RNA was isolated from modified cells and qRT-PCR with Taqmanprimer-probe sets was used to quantify expression. Expression isnormalized to the Beta-Actin housekeeping gene and each sample isnormalized to the GFP control sample (cells transfected with only GFP).Error bars represent standard error of the mean of two to six biologicalreplicates.

FIG. 14 shows deletion rates in cells transfected with indicated gRNApairs and Cas9 as measured by droplet digital PCR (ddPCR). % deletionwas calculated by dividing the number of positive droplets in deletionassay by the number of positive droplets in a control assay. Threebiological replicates are shown for two different gRNA pairs.

FIG. 15 shows deletion rates in 293T cells transfected with exemplaryAAV expression plasmids. pSS10 encodes EFS-driven saCas9 without gRNA.pSS15 and pSS17 encode EFS-driven saCas9 and one U6-driven gRNA,CEP290-64 and CEP290-323 respectively. pSS11 encodes EFS-driven saCas9and two U6-driven gRNAs, CEP290-64 and CEP290-323 in the same vector.Deletion PCR were performed with gDNA exacted from 293T cells posttransfection. The size of the PCR amplicons indicates the presence orabsence of deletion events, and the deletion ratio was calculated.

FIG. 16 shows the composition of structural proteins in AAV2 viral prepsexpressing Cas9. Reference AAV2 vectors (lanes 1 & 2) were obtained fromVector Core at University of North Carolina, Chapel Hill. AAV2-CMV-GFP(lane 3) and AAV2-CMV-saCas9-minpA (lane4) were packaged and purifiedwith “Triple Transfection Protocol” followed by CsClultracentrifugation. Titers were obtained by quantitative PCR withprimers annealing to the ITR structures on these vectors. Viral prepswere denatured and probed with B1 antibody on Western Blots todemonstrate three structural proteins composing AAV2, VP1, VP2, and VP3respectively.

FIG. 17 depicts the deletion rates in 293T cells transduced with AAVviral vectors at MOI of 1000 viral genome (vg) per cell and 10,000 vgper cell. AAV2 viral vectors were produced with “Triple TransfectionProtocol” using pHelper, pRep2Cap2, pSS8 encoding gRNAs CEP290-64 andCEP290-323, and CMV-driven saCas9. Viral preps were titered with primersannealing to ITRs on pSS8. 6 days post transduction, gDNA were extractedfrom 293T cells. Deletion PCR was carried out on the CEP290 locus, anddeletion rates were calculated based on the predicted amplicons. Westernblotting was carried out to show the AAV-mediated saCas9 expression in293T cells (primary antibody: anti-Flag, M2; loading control:anti-alphaTubulin).

FIG. 18A-18B depicts additional exemplary structures of unimoleculargRNA molecules. FIG. 18A (SEQ ID NO: 45) shows an exemplary structure ofa unimolecular gRNA molecule derived in part from S. pyogenes as aduplexed structure. FIG. 18B (SEQ ID NO: 2779) shows an exemplarystructure of a unimolecular gRNA molecule derived in part from S. aureusas a duplexed structure.

FIGS. 19A-19G depicts the nucleotide sequence of an exemplaryrecombinant AAV genome containing a CMV promoter. Various components ofthe recombinant AAV genome are also indicated. N=A, T, G or C. Thenumber of N residues can vary, e.g., from 16 to 26 nucleotides. Upperstand: 5′→3′ (SEQ ID NO: 428); lower stand: 3′→5′ SEQ ID NO: 445).

FIGS. 20A-20F depicts the nucleotide sequence of an exemplaryrecombinant AAV genome containing an EFS promoter. Various components ofthe recombinant AAV genome are also indicated. N=A, T, G or C. Thenumber of N residues can vary, e.g., from 16 to 26 nucleotides. Upperstand: 5′→3′ (SEQ ID NO: 429); lower stand: 3′→5′ (SEQ ID NO: 446).

FIGS. 21A-21F depicts the nucleotide sequence of an exemplaryrecombinant AAV genome containing a CRK1 promoter. Various components ofthe recombinant AAV genome are also indicated. N=A, T, G or C. Thenumber of N residues can vary, e.g., from 16 to 26 nucleotides. Upperstand: 5′→3′ (SEQ ID NO: 430); lower stand: 3′→5′ (SEQ ID NO: 447).

FIGS. 22A-22F depicts the nucleotide sequence of an exemplaryrecombinant AAV genome containing a CRX promoter. Various components ofthe recombinant AAV genome are also indicated. N=A, T, G or C. Thenumber of N residues can vary, e.g., from 16 to 26 nucleotides. Upperstand: 5′→3′ (SEQ ID NO: 431); lower stand: 3′→5′ (SEQ ID NO: 448).

FIGS. 23A-23F depicts the nucleotide sequence of an exemplaryrecombinant AAV genome containing a NRL promoter. Various components ofthe recombinant AAV genome are also indicated. N=A, T, G or C. Thenumber of N residues can vary, e.g., from 16 to 26 nucleotides. Upperstand: 5′→3′ (SEQ ID NO: 432); lower stand: 3′→5′ (SEQ ID NO: 449).

FIGS. 24A-24F depicts the nucleotide sequence of an exemplaryrecombinant AAV genome containing a NRL promoter. Various components ofthe recombinant AAV genome are also indicated. N=A, T, G or C. Thenumber of N residues can vary, e.g., from 16 to 26 nucleotides. Upperstand: 5′→3′ (SEQ ID NO: 433); lower stand: 3′→5′ (SEQ ID NO: 450).

FIGS. 25A-D include schematic depictions of exemplary AAV viral genomeaccording to certain embodiments of the disclosure. FIG. 25A shows anAAV genome for use in altering a CEP290 target position which encodes,inter alia, two guide RNAs having specific targeting domains selectedfrom SEQ ID NOs: 389-391, 388, 392, and 394 and an S. aureus Cas9. Incertain embodiments, the AAV genome having the configuration illustratedin FIG. 25A may comprise the sequence set forth in SEQ ID NO: 2802. Incertain of those embodiments, the genome having the configurationillustrated in FIG. 25A may comprise the sequence set forth in SEQ IDNO: 2803. FIG. 25B shows an AAV genome that may be used for a variety ofapplications, including without limitation the alteration of the CEP290target position, encoding two guide RNAs comprising the sequences of SEQID NOs: 2785 and 2787 and an S. aureus Cas9. FIG. 25C shows an AAVgenome encoding one or two guide RNAs, each driven by a U6 promoter, andan S. aureus Cas9. In the figure, N may be 1 or two. FIG. 25D shows afurther annotated version of FIG. 25A, illustrating an AAV genome foruse in altering a CEP290 target position which encodes the targetingdomains from SEQ ID NOs: 389 and 388 and an S. aureus Cas9. In certainembodiments, the AAV genome having the configuration illustrated in FIG.25D may comprise the sequence set forth in SEQ ID NO: 2802. In certainof those embodiments, the genome having the configuration illustrated inFIG. 25D may comprise the sequence set forth in SEQ ID NO: 2803.

FIG. 26 illustrates the genome editing strategy implemented in certainembodiments of this disclosure.

FIG. 27A shows a photomicrograph of a mouse retinal explant on a supportmatrix; retinal tissue is indicated by the arrow. FIG. 27B shows afluorescence micrograph from a histological section of a mouse retinalexplant illustrating AAV transduction of cells in multiple retinallayers with a GFP reporter. FIG. 27C shows a micrograph from ahistological section of a primate retinal tissue treated with vehicle.FIG. 27D shows a micrograph from a histological section of a primateretinal tissue treated with AAV5 vector encoding S. aureus Cas9 operablylinked to the photoreceptor-specific hGRK1 promoter. Dark staining inthe outer nuclear layer (ONL) indicates that cells were successfullytransduced with AAV and express Cas9.

FIG. 28A and FIG. 28B show expression of Cas9 mRNA and gRNA,respectively, normalized to GAPDH mRNA expression. UT denotes untreated;GRK1-Cas refers to a vector in which Cas9 expression is driven by thephotoreceptor-specific hGRK1 promoter; dCMV-Cas and EFS-Cas similarlyrefer to vectors in which Cas9 expression is driven by the dCMV promoteror the EFS promoter. Conditions in which gRNAs are included in thevector are denoted by the bar captioned “with gRNA.” Light and dark barsdepict separate experimental replicates.

FIG. 29 summarizes the edits observed in mouse retinal explants 7 daysafter transduction with AAV5-mCEPgRNAs-Cas9. Edits were binned into oneof three categories: no edit, indel at one of two guide sites, anddeletion of sequence between the guide sites. Each bar graph depicts theobserved edits as a percentage of sequence reads from individualexplants transduced with AAV vectors in which Cas9 was driven by thepromoter listed (hGRK1, CMV or EFS).

FIG. 30 summarizes the edits observed in the CEP290 gene in retinalpunch samples obtained from cynomolgus monkeys treated with AAV vectorsencoding genome editing systems according to the present disclosure.

FIG. 31A depicts a reporter construct that was used to assess the effectof certain editing outcomes, including inversions and deletions, on theIVS26 splicing defect. FIG. 31B depicts the relative levels of GFPreporter expression in WT, IVS26, deletion and inversion conditions,normalized to mCherry expression.

FIG. 32 summarizes the productive CEP290 edits observed in human retinalexplants 14 or 28 days after transduction with AAV vectors in which Cas9was driven by the promoter listed (hGRK1 or CMV).

DETAILED DESCRIPTION Definitions

Unless otherwise specified, each of the following terms has the meaningset forth in this section.

The indefinite articles “a” and “an” denote at least one of theassociated noun, and are used interchangeably with the terms “at leastone” and “one or more.” For example, the phrase “a module” means atleast one module, or one or more modules.

The conjunctions “or” and “and/or” are used interchangeably.

“Domain” as used herein is used to describe segments of a protein ornucleic acid. Unless otherwise indicated, a domain is not required tohave any specific functional property.

An “indel” is an insertion and/or deletion in a nucleic acid sequence.An indel may be the product of the repair of a DNA double strand break,such as a double strand break formed by a genome editing system of thepresent disclosure. An indel is most commonly formed when a break isrepaired by an “error prone” repair pathway such as the NHEJ pathwaydescribed below. Indels are typically assessed by sequencing (mostcommonly by “next-gen” or “sequencing-by-synthesis” methods, thoughSanger sequencing may still be used) and are quantified by the relativefrequency of numerical changes (e.g., ±1, ±2 or more bases) at a site ofinterest among all sequencing reads. DNA samples for sequencing can beprepared by a variety of methods known in the art, and may involve theamplification of sites of interest by polymerase chain reaction (PCR) orthe capture of DNA ends generated by double strand breaks, as in theGUIDEseq process described in Tsai 2016 (incorporated by referenceherein). Other sample preparation methods are known in the art. Indelsmay also be assessed by other methods, including in situ hybridizationmethods such as the FiberComb™ system commercialized by Genomic Vision(Bagneux, France), and other methods known in the art.

“CEP290 target position” and “CEP290 target site” are usedinterchangeably herein to refer to a nucleotide or nucleotides in ornear the CEP290 gene that are targeted for alteration using the methodsdescribed herein. In certain embodiments, a mutation at one or more ofthese nucleotides is associated with a CEP290 associated disease. Theterms “CEP290 target position” and “CEP290 target site” are also usedherein to refer to these mutations. For example, the IVS26 mutation isone non-limiting embodiment of a CEP290 target position/target site.

Calculations of homology or sequence identity between two sequences (theterms are used interchangeably herein) are performed as follows. Thesequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleicacid sequence for optimal alignment and non-homologous sequences can bedisregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences.

“Governing gRNA molecule” as used herein refers to a gRNA molecule thatcomprises a targeting domain that is complementary to a target domain ona nucleic acid that comprises a sequence that encodes a component of theCRISPR/Cas system that is introduced into a cell or subject. A governinggRNA does not target an endogenous cell or subject sequence. In anembodiment, a governing gRNA molecule comprises a targeting domain thatis complementary with a target sequence on: (a) a nucleic acid thatencodes a Cas9 molecule; (b) a nucleic acid that encodes a gRNA whichcomprises a targeting domain that targets the CEP290 gene (a target genegRNA); or on more than one nucleic acid that encodes a CRISPR/Cascomponent, e.g., both (a) and (b). In an embodiment, a nucleic acidmolecule that encodes a CRISPR/Cas component, e.g., that encodes a Cas9molecule or a target gene gRNA, comprises more than one target domainthat is complementary with a governing gRNA targeting domain. While notwishing to be bound by theory, it is believed that a governing gRNAmolecule complexes with a Cas9 molecule and results in Cas9 mediatedinactivation of the targeted nucleic acid, e.g., by cleavage or bybinding to the nucleic acid, and results in cessation or reduction ofthe production of a CRISPR/Cas system component. In an embodiment, theCas9 molecule forms two complexes: a complex comprising a Cas9 moleculewith a target gene gRNA, which complex will alter the CEP290 gene; and acomplex comprising a Cas9 molecule with a governing gRNA molecule, whichcomplex will act to prevent further production of a CRISPR/Cas systemcomponent, e.g., a Cas9 molecule or a target gene gRNA molecule. In anembodiment, a governing gRNA molecule/Cas9 molecule complex binds to orpromotes cleavage of a control region sequence, e.g., a promoter,operably linked to a sequence that encodes a Cas9 molecule, a sequencethat encodes a transcribed region, an exon, or an intron, for the Cas9molecule. In an embodiment, a governing gRNA molecule/Cas9 moleculecomplex binds to or promotes cleavage of a control region sequence,e.g., a promoter, operably linked to a gRNA molecule, or a sequence thatencodes the gRNA molecule. In an embodiment, the governing gRNA, e.g., aCas9-targeting governing gRNA molecule, or a target gene gRNA-targetinggoverning gRNA molecule, limits the effect of the Cas9 molecule/targetgene gRNA molecule complex-mediated gene targeting. In an embodiment, agoverning gRNA places temporal, level of expression, or other limits, onactivity of the Cas9 molecule/target gene gRNA molecule complex. In anembodiment, a governing gRNA reduces off-target or other unwantedactivity. In an embodiment, a governing gRNA molecule inhibits, e.g.,entirely or substantially entirely inhibits, the production of acomponent of the Cas9 system and thereby limits, or governs, itsactivity.

“Modulator” as used herein refers to an entity, e.g., a drug that canalter the activity (e.g., enzymatic activity, transcriptional activity,or translational activity), amount, distribution, or structure of asubject molecule or genetic sequence. In an embodiment, modulationcomprises cleavage, e.g., breaking of a covalent or non-covalent bond,or the forming of a covalent or non-covalent bond, e.g., the attachmentof a moiety, to the subject molecule. In an embodiment, a modulatoralters the, three dimensional, secondary, tertiary, or quaternarystructure, of a subject molecule. A modulator can increase, decrease,initiate, or eliminate a subject activity.

“Large molecule” as used herein refers to a molecule having a molecularweight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100kD. Large molecules include proteins, polypeptides, nucleic acids,biologics, and carbohydrates.

“Polypeptide” as used herein refers to a polymer of amino acids havingless than 100 amino acid residues. In an embodiment, it has less than50, 20, or 10 amino acid residues.

“Non-homologous end joining” or “NHEJ”, as used herein, refers toligation mediated repair and/or non-template mediated repair including,e.g., canonical NHEJ (cNHEJ), alternative NHEJ (altNHEJ),microhomology-mediated end joining (MMEJ), single-strand annealing(SSA), and synthesis-dependent microhomology-mediated end joining(SD-MMEJ).

“Reference molecule”, e.g., a reference Cas9 molecule or reference gRNA,as used herein refers to a molecule to which a subject molecule, e.g., asubject Cas9 molecule of subject gRNA molecule, e.g., a modified orcandidate Cas9 molecule is compared. For example, a Cas9 molecule can becharacterized as having no more than 10% of the nuclease activity of areference Cas9 molecule. Examples of reference Cas9 molecules includenaturally occurring unmodified Cas9 molecules, e.g., a naturallyoccurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, S.aureus, or S. thermophilus. In an embodiment, the reference Cas9molecule is the naturally occurring Cas9 molecule having the closestsequence identity or homology with the Cas9 molecule to which it isbeing compared. In an embodiment, the reference Cas9 molecule is asequence, e.g., a naturally occurring or known sequence, which is theparental form on which a change, e.g., a mutation has been made.

“Replacement”, or “replaced”, as used herein with reference to amodification of a molecule does not require a process limitation butmerely indicates that the replacement entity is present.

“Small molecule” as used herein refers to a compound having a molecularweight less than about 2 kD, e.g., less than about 2 kD, less than about1.5 kD, less than about 1 kD, or less than about 0.75 kD.

“Subject” as used herein means a human, mouse, or non-human primate. Ahuman subject can be any age (e.g., an infant, child, young adult, oradult), and may suffer from a disease, or may be in need of alterationof a gene.

“Treat,” “treating,” and “treatment” as used herein mean the treatmentof a disease in a subject (e.g., a human subject), including one or moreof inhibiting the disease, i.e., arresting or preventing its developmentor progression; relieving the disease, i.e., causing regression of thedisease state; relieving one or more symptoms of the disease; and curingthe disease.

“Prevent,” “preventing,” and “prevention” as used herein means theprevention of a disease in a subject, e.g., in a human, including (a)avoiding or precluding the disease; (b) affecting the predispositiontoward the disease; (c) preventing or delaying the onset of at least onesymptom of the disease.

The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”,“nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide”as used herein refer to a series of nucleotide bases (also called“nucleotides”) in DNA and RNA, and mean any chain of two or morenucleotides. The polynucleotides can be chimeric mixtures or derivativesor modified versions thereof, single-stranded or double-stranded. Theoligonucleotide can be modified at the base moiety, sugar moiety, orphosphate backbone, for example, to improve stability of the molecule,its hybridization parameters, etc. A nucleotide sequence typicallycarries genetic information, including the information used by cellularmachinery to make proteins and enzymes. These terms include double- orsingle-stranded genomic DNA, RNA, any synthetic and geneticallymanipulated polynucleotide, and both sense and antisensepolynucleotides. This also includes nucleic acids containing modifiedbases.

“X” as used herein in the context of an amino acid sequence, refers toany amino acid (e.g., any of the twenty natural amino acids) unlessotherwise specified.

Conventional IUPAC notation is used in nucleotide sequences presentedherein, as shown in Table 1, below (see also Cornish-Bowden 1985,incorporated by reference herein). It should be noted, however, that “T”denotes “Thymine or Uracil” insofar as a given sequence (such as a gRNAsequence) may be encoded by either DNA or RNA.

TABLE 1 IUPAC nucleic acid notation Character Base A Adenine T Thymine GGuanine C Cytosine U Uracil K G or T/U M A or C R A or G Y C or T/U S Cor G W A or T/U B C, G, or T/U V A, C, or G H A, C, or T/U D A, G, orT/U N A, C, G, or T/U

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein to refer to a sequential chain of amino acidslinked together via peptide bonds. The terms include individualproteins, groups or complexes of proteins that associate together, aswell as fragments, variants, derivatives and analogs of such proteins.Peptide sequences are presented using conventional notation, beginningwith the amino or N-terminus on the left, and proceeding to the carboxylor C-terminus on the right. Standard one-letter or three-letterabbreviations may be used.

Methods of Altering CEP290

CEP290 encodes a centrosomal protein that plays a role in centrosome andcilia development. The CEP290 gene is involved in forming cilia aroundcells, particularly in the photoreceptors at the back of the retina,which are needed to detect light and color.

Disclosed herein are methods and compositions for altering the LCA10target position in the CEP290 gene. LCA10 target position can be altered(e.g., corrected) by gene editing, e.g., using CRISPR-Cas9 mediatedmethods. The alteration (e.g., correction) of the mutant CEP290 gene canbe mediated by any mechanism. Exemplary mechanisms that can beassociated with the alteration (e.g., correction) of the mutant CEP290gene include, but are not limited to, non-homologous end joining (e.g.,classical or alternative), microhomology-mediated end joining (MMEJ),homology-directed repair (e.g., endogenous donor template mediated),SDSA (synthesis dependent strand annealing), single strand annealing orsingle strand invasion. Methods described herein introduce one or morebreaks near the site of the LCA target position (e.g., c.2991+1655A toG) in at least one allele of the CEP290 gene. In an embodiment, the oneor more breaks are repaired by NHEJ. During repair of the one or morebreaks, DNA sequences are inserted and/or deleted resulting in the lossor destruction of the cryptic splice site resulting from the mutation atthe LCA10 target position (e.g., c.2991+1655A to G). The method caninclude acquiring knowledge of the mutation carried by the subject,e.g., by sequencing the appropriate portion of the CEP290 gene.

Altering the LCA10 target position refers to (1) break-inducedintroduction of an indel (also referred to herein as NHEJ-mediatedintroduction of an indel) in close proximity to or including a LCA10target position (e.g., c.2991+1655A to G), or (2) break-induced deletion(also referred to herein as NHEJ-mediated deletion) of genomic sequenceincluding the mutation at a LCA10 target position (e.g., c.2991+1655A toG). Both approaches give rise to the loss or destruction of the crypticsplice site.

In an embodiment, the method comprises introducing a break-induced indelin close proximity to or including the LCA10 target position (e.g.,c.2991+1655A to G). As described herein, in one embodiment, the methodcomprises the introduction of a double strand break sufficiently closeto (e.g., either 5′ or 3′ to) the LCA10 target position, e.g.,c.2991+1655A to G, such that the break-induced indel could be reasonablyexpected to span the mutation. A single gRNAs, e.g., unimolecular (orchimeric) or modular gRNA molecules, is configured to position a doublestrand break sufficiently close to the LCA10 target position in theCEP290 gene. In an embodiment, the break is positioned to avoid unwantedtarget chromosome elements, such as repeat elements, e.g., an Alurepeat. The double strand break may be positioned within 40 nucleotides(e.g., within 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25, 30, 35 or40 nucleotides) upstream of the LCA10 target position, or within 40nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15, 16, 17, 18, 19, 20, 25,30, 35 or 40 nucleotides) downstream of the LCA10 target position (seeFIG. 9). While not wishing to be bound by theory, in an embodiment, itis believed that NHEJ-mediated repair of the double strand break allowsfor the NHEJ-mediated introduction of an indel in close proximity to orincluding the LCA10 target position.

In another embodiment, the method comprises the introduction of a pairof single strand breaks sufficiently close to (either 5′ or 3′ to,respectively) the mutation at the LCA10 target position (e.g.,c.2991+1655A to G) such that the break-induced indel could be reasonablyexpected to span the mutation. Two gRNAs, e.g., unimolecular (orchimeric) or modular gRNA molecules, are configured to position the twosingle strand breaks sufficiently close to the LCA10 target position inthe CEP290 gene. In an embodiment, the breaks are positioned to avoidunwanted target chromosome elements, such as repeat elements, e.g., anAlu repeat. In an embodiment, the pair of single strand breaks ispositioned within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15,16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) upstream of the LCA10target position, or within 40 nucleotides (e.g., within 1, 2, 3, 4, 5,10, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) downstream ofthe LCA10 target position (see FIG. 9). While not wishing to be bound bytheory, in an embodiment, it is believed that NHEJ mediated repair ofthe pair of single strand breaks allows for the NHEJ-mediatedintroduction of an indel in close proximity to or including the LCA10target position. In an embodiment, the pair of single strand breaks maybe accompanied by an additional double strand break, positioned by athird gRNA molecule, as is discussed below. In another embodiment, thepair of single strand breaks may be accompanied by two additional singlestrand breaks positioned by a third gRNA molecule and a fourth gRNAmolecule, as is discussed below.

In an embodiment, the method comprises introducing a break-induceddeletion of genomic sequence including the mutation at the LCA10 targetposition (e.g., c.2991+1655A to G). As described herein, in oneembodiment, the method comprises the introduction of two double strandbreaks—one 5′ and the other 3′ to (i.e., flanking) the LCA10 targetposition. Two gRNAs, e.g., unimolecular (or chimeric) or modular gRNAmolecules, are configured to position the two double strand breaks onopposite sides of the LCA10 target position in the CEP290 gene. In anembodiment, the first double strand break is positioned upstream of theLCA10 target position within intron 26 (e.g., within 1654 nucleotides),and the second double strand break is positioned downstream of the LCA10target position within intron 26 (e.g., within 4183 nucleotides) (seeFIG. 10). In an embodiment, the breaks (i.e., the two double strandbreaks) are positioned to avoid unwanted target chromosome elements,such as repeat elements, e.g., an Alu repeat, or the endogenous CEP290splice sites.

The first double strand break may be positioned as follows:

-   -   (1) upstream of the 5′ end of the Alu repeat in intron 26,    -   (2) between the 3′ end of the Alu repeat and the LCA10 target        position in intron 26, or    -   (3) within the Alu repeat provided that a sufficient length of        the gRNA fall outside of the repeat so as to avoid binding to        other Alu repeats in the genome, and the second double strand        break to be paired with the first double strand break may be        positioned downstream of the LCA10 target position in intron 26.

For example, the first double strand break may be positioned:

-   -   (1) within 1162 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (2) within 1000 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (3) within 900 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (4) within 800 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (5) within 700 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (6) within 600 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (7) within 500 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (8) within 400 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (9) within 300 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (10) within 200 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (11) within 100 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (12) within 50 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (13) within the Alu repeat provided that a sufficient length of        the gRNA falls outside of the repeat so as to avoid binding to        other Alu repeats in the genome,    -   (14) within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15,        16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) upstream of        the LCA10 target position, or    -   (15) within 17 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15,        16 or 17 nucleotides) upstream of the LCA10 target position,        and the second double strand breaks to be paired with the first        double strand break may be positioned:    -   (1) within 4183 nucleotides downstream of the LCA10 target        position,    -   (2) within 4000 nucleotides downstream of the LCA10 target        position,    -   (3) within 3000 nucleotides downstream of the LCA10 target        position,    -   (4) within 2000 nucleotides downstream of the LCA10 target        position,    -   (5) within 1000 nucleotides downstream of the LCA10 target        position,    -   (6) within 700 nucleotides downstream of the LCA10 target        position,    -   (7) within 500 nucleotides downstream of the LCA10 target        position,    -   (8) within 300 nucleotides downstream of the LCA10 target        position,    -   (9) within 100 nucleotides downstream of the LCA10 target        position,    -   (10) within 60 nucleotides downstream of the LCA10 target        position, or    -   (11) within 40 (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or        40 nucleotides) nucleotides downstream of the LCA10 target        position.

While not wishing to be bound by theory, in an embodiment, it isbelieved that the two double strand breaks allow for break-induceddeletion of genomic sequence including the mutation at the LCA10 targetposition in the CEP290 gene.

The method also comprises the introduction of two sets of breaks, e.g.,one double strand break (either 5′ or 3′ to the mutation at the LCA10target position, e.g., c.2991+1655A to G) and a pair of single strandbreaks (on the other side of the LCA10 target position opposite from thedouble strand break) such that the two sets of breaks are positioned toflank the LCA10 target position. Three gRNAs, e.g., unimolecular (orchimeric) or modular gRNA molecules, are configured to position the onedouble strand break and the pair of single strand breaks on oppositesides of the LCA10 target position in the CEP290 gene. In an embodiment,the first set of breaks (either the double strand break or the pair ofsingle strand breaks) is positioned upstream of the LCA10 targetposition within intron 26 (e.g., within 1654 nucleotides), and thesecond set of breaks (either the double strand break or the pair ofsingle strand breaks) are positioned downstream of the LCA10 targetposition within intron 26 (e.g., within 4183 nucleotides) (see FIG. 10).In an embodiment, the two sets of breaks (i.e., the double strand breakand the pair of single strand breaks) are positioned to avoid unwantedtarget chromosome elements, such as repeat elements, e.g., an Alurepeat, or the endogenous CEP290 splice sites.

The first set of breaks (either the double strand break or the pair ofsingle strand breaks) may be positioned:

-   -   (1) upstream of the 5′ end of the Alu repeat in intron 26,    -   (2) between the 3′ end of the Alu repeat and the LCA10 target        position in intron 26, or    -   (3) within the Alu repeat provided that a sufficient length of        the gRNA falls outside of the repeat so as to avoid binding to        other Alu repeats in the genome, and the second set of breaks to        be paired with the first set of breaks (either the double strand        break or the pair of single strand breaks) may be positioned        downstream of the LCA10 target position in intron 26.

For example, the first set of breaks (either the double strand break orthe pair of single strand breaks) may be positioned:

-   -   (1) within 1162 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (2) within 1000 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (3) within 900 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (4) within 800 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (5) within 700 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (6) within 600 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (7) within 500 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (8) within 400 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (9) within 300 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (10) within 200 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (11) within 100 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (12) within 50 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (13) within the Alu repeat provided that a sufficient length of        the gRNA falls outside of the repeat so as to avoid binding to        other Alu repeats in the genome,    -   (14) within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15,        16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) upstream of        the LCA10 target position, or    -   (15) within 17 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15,        16 or 17 nucleotides) upstream of the LCA10 target position,        and the second set of breaks to be paired with the first set of        breaks (either the double strand break or the pair of single        strand breaks) may be positioned:    -   (1) within 4183 nucleotides downstream of the LCA10 target        position,    -   (2) within 4000 nucleotides downstream of the LCA10 target        position,    -   (3) within 3000 nucleotides downstream of the LCA10 target        position,    -   (4) within 2000 nucleotides downstream of the LCA10 target        position,    -   (5) within 1000 nucleotides downstream of the LCA10 target        position,    -   (6) within 700 nucleotides downstream of the LCA10 target        position,    -   (7) within 500 nucleotides downstream of the LCA10 target        position,    -   (8) within 300 nucleotides downstream of the LCA10 target        position,    -   (9) within 100 nucleotides downstream of the LCA10 target        position,    -   (10) within 60 nucleotides downstream of the LCA10 target        position, or    -   (11) within 40 (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or        40 nucleotides) nucleotides downstream of the LCA10 target        position.

While not wishing to be bound by theory, it is believed that the twosets of breaks (either the double strand break or the pair of singlestrand breaks) allow for break-induced deletion of genomic sequenceincluding the mutation at the LCA10 target position in the CEP290 gene.

The method also comprises the introduction of two sets of breaks, e.g.,two pairs of single strand breaks, wherein the two sets ofsingle-stranded breaks are positioned to flank the LCA10 targetposition. In an embodiment, the first set of breaks (e.g., the firstpair of single strand breaks) is 5′ to the mutation at the LCA10 targetposition (e.g., c.2991+1655A to G) and the second set of breaks (e.g.,the second pair of single strand breaks) is 3′ to the mutation at theLCA10 target position. Four gRNAs, e.g., unimolecular (or chimeric) ormodular gRNA molecules, are configured to position the two pairs ofsingle strand breaks on opposite sides of the LCA10 target position inthe CEP290 gene. In an embodiment, the first set of breaks (e.g., thefirst pair of single strand breaks) is positioned upstream of the LCA10target position within intron 26 (e.g., within 1654 nucleotides), andthe second set of breaks (e.g., the second pair of single strand breaks)is positioned downstream of the LCA10 target position within intron 26(e.g., within 4183 nucleotides) (see FIG. 10). In an embodiment, the twosets of breaks (i.e., the two pairs of single strand breaks) arepositioned to avoid unwanted target chromosome elements, such as repeatelements, e.g., an Alu repeat, or the endogenous CEP290 splice sites.

The first set of breaks (e.g., the first pair of single strand breaks)may be positioned:

-   -   (1) upstream of the 5′ end of the Alu repeat in intron 26,    -   (2) between the 3′ end of the Alu repeat and the LCA10 target        position in intron 26, or    -   (3) within the Alu repeat provided that a sufficient length of        the gRNA falls outside of the repeat so as to avoid binding to        other Alu repeats in the genome,        and the second set of breaks to be paired with the first set of        breaks (e.g., the second pair of single strand breaks) may be        positioned downstream of the LCA10 target position in intron 26.

For example, the first set of breaks (e.g., the first pair of singlestrand breaks) may be positioned:

-   -   (1) within 1162 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (2) within 1000 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (3) within 900 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (4) within 800 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (5) within 700 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (6) within 600 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (7) within 500 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (8) within 400 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (9) within 300 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (10) within 200 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (11) within 100 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (12) within 50 nucleotides upstream of the 5′ end of the Alu        repeat,    -   (13) within the Alu repeat provided that a sufficient length of        the gRNA falls outside of the repeat so as to avoid binding to        other Alu repeats in the genome,    -   (14) within 40 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15,        16, 17, 18, 19, 20, 25, 30, 35 or 40 nucleotides) upstream of        the LCA10 target position, or    -   (15) within 17 nucleotides (e.g., within 1, 2, 3, 4, 5, 10, 15,        16 or 17 nucleotides) upstream of the LCA10 target position,        and the second set of breaks to be paired with the first set of        breaks (e.g., the second pair of single strand breaks) may be        positioned:    -   (1) within 4183 nucleotides downstream of the LCA10 target        position,    -   (2) within 4000 nucleotides downstream of the LCA10 target        position,    -   (3) within 3000 nucleotides downstream of the LCA10 target        position,    -   (4) within 2000 nucleotides downstream of the LCA10 target        position,    -   (5) within 1000 nucleotides downstream of the LCA10 target        position,    -   (6) within 700 nucleotides downstream of the LCA10 target        position,    -   (7) within 500 nucleotides downstream of the LCA10 target        position,    -   (8) within 300 nucleotides downstream of the LCA10 target        position,    -   (9) within 100 nucleotides downstream of the LCA10 target        position,    -   (10) within 60 nucleotides downstream of the LCA10 target        position, or    -   (11) within 40 (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35 or        40 nucleotides) nucleotides downstream of the LCA10 target        position.

While not wishing to be bound by theory, it is believed that the twosets of breaks (e.g., the two pairs of single strand breaks) allow forbreak-induced deletion of genomic sequence including the mutation at theLCA10 target position in the CEP290 gene.

Methods of Treating or Preventing LCA10

Described herein are methods for treating or delaying the onset orprogression of Leber's Congenital Amaurosis 10 (LCA10) caused by ac.2991+1655 A to G (adenine to guanine) mutation in the CEP290 gene. Thedisclosed methods for treating or delaying the onset or progression ofLCA10 alter the CEP290 gene by genome editing using a gRNA targeting theLCA10 target position and a Cas9 enzyme. Details on gRNAs targeting theLCA10 target position and Cas9 enzymes are provided below.

In an embodiment, treatment is initiated prior to onset of the disease.

In an embodiment, treatment is initiated after onset of the disease.

In an embodiment, treatment is initiated prior to loss of visual acuityand/or sensitivity to glare.

In an embodiment, treatment is initiated at onset of loss of visualacuity.

In an embodiment, treatment is initiated after onset of loss of visualacuity and/or sensitivity to glare.

In an embodiment, treatment is initiated in utero.

In an embodiment, treatment is initiated after birth.

In an embodiment, treatment is initiated prior to the age of 1.

In an embodiment, treatment is initiated prior to the age of 2.

In an embodiment, treatment is initiated prior to the age of 5.

In an embodiment, treatment is initiated prior to the age of 10.

In an embodiment, treatment is initiated prior to the age of 15.

In an embodiment, treatment is initiated prior to the age of 20.

A subject's vision can evaluated, e.g., prior to treatment, or aftertreatment, e.g., to monitor the progress of the treatment. In anembodiment, the subject's vision is evaluated prior to treatment, e.g.,to determine the need for treatment. In an embodiment, the subject'svision is evaluated after treatment has been initiated, e.g., to accessthe effectiveness of the treatment. Vision can be evaluated by one ormore of: evaluating changes in function relative to the contralateraleye, e.g., by utilizing retinal analytical techniques; by evaluatingmean, median and distribution of change in best corrected visual acuity(BCVA); evaluation by Optical Coherence Tomography; evaluation ofchanges in visual field using perimetry; evaluation by full-fieldelectroretinography (ERG); evaluation by slit lamp examination;evaluation of intraocular pressure; evaluation of autofluorescence,evaluation with fundoscopy; evaluation with fundus photography;evaluation with fluorescein angiography (FA); or evaluation of visualfield sensitivity (FFST).

In an embodiment, a subject's vision may be assessed by measuring thesubject's mobility, e.g., the subject's ability to maneuver in space.

In an embodiment, treatment is initiated in a subject who has testedpositive for a mutation in the CEP290 gene, e.g., prior to disease onsetor in the earliest stages of disease.

In an embodiment, a subject has a family member that has been diagnosedwith LCA10. For example, the subject has a family member that has beendiagnosed with LCA10, and the subject demonstrates a symptom or sign ofthe disease or has been found to have a mutation in the CEP290 gene.

In an embodiment, a cell (e.g., a retinal cell, e.g., a photoreceptorcell) from a subject suffering from or likely to develop LCA10 istreated ex vivo. In an embodiment, the cell is removed from the subject,altered as described herein, and introduced into, e.g., returned to, thesubject.

In an embodiment, a cell (e.g., a retinal cell, e.g., a photoreceptorcell) altered to correct a mutation in the LCA10 target position isintroduced into the subject.

In an embodiment, the cell is a retinal cell (e.g., retinal pigmentepithelium cell), a photoreceptor cell, a horizontal cell, a bipolarcell, an amacrine cell, or a ganglion cell. In an embodiment, it iscontemplated herein that a population of cells (e.g., a population ofretinal cells, e.g., a population of photoreceptor cells) from a subjectmay be contacted ex vivo to alter a mutation in CEP290, e.g., a2991+1655 A to G. In an embodiment, such cells are introduced to thesubject's body to prevent or treat LCA10.

In an embodiment, the population of cells are a population of retinalcells (e.g., retinal pigment epithelium cells), photoreceptor cells,horizontal cells, bipolar cells, amacrine cells, ganglion cells, or acombination thereof.

In an embodiment, the method described herein comprises delivery of gRNAor other components described herein, e.g., a Cas9 molecule, by one ormore AAV vectors, e.g., one or more AAV vectors described herein.

I. Genome Editing Systems

The term “genome editing system” refers to any system having RNA-guidedDNA editing activity. Genome editing systems of the present disclosureinclude at least two components adapted from naturally occurring CRISPRsystems: a gRNA and an RNA-guided nuclease. These two components form acomplex that is capable of associating with a specific nucleic acidsequence in a cell and editing the DNA in or around that nucleic acidsequence, for example by making one or more of a single-strand break (anSSB or nick), a double-strand break (a DSB) and/or a base substitution.

Naturally occurring CRISPR systems are organized evolutionarily into twoclasses and five types (Makarova 2011, incorporated by referenceherein), and while genome editing systems of the present disclosure mayadapt components of any type or class of naturally occurring CRISPRsystem, the embodiments presented herein are generally adapted fromClass 2, and type II or V CRISPR systems. Class 2 systems, whichencompass types II and V, are characterized by relatively large,multidomain RNA-guided nuclease proteins (e.g., Cas9 or Cpf1) that formribonucleoprotein (RNP) complexes with gRNAs. gRNAs, which are discussedin greater detail below, can include single crRNAs in the case of Cpf1or duplexed crRNAs and tracrRNAs in the case of Cas9. RNP complexes, inturn, associate with (i.e., target) and cleave specific locicomplementary to a targeting (or spacer) sequence of the crRNA. Genomeediting systems according to the present disclosure similarly target andedit cellular DNA sequences. but differ significantly from CRISPRsystems occurring in nature. For example, the unimolecular gRNAsdescribed herein do not occur in nature, and both gRNAs and RNA-guidednucleases according to this disclosure can incorporate any number ofnon-naturally occurring modifications.

Genome editing systems can be implemented in a variety of ways, anddifferent implementations may be suitable for any particularapplication. For example, a genome editing system is implemented, incertain embodiments, as a protein/RNA complex (a ribonucleoprotein, orRNP), which can be included in a pharmaceutical composition thatoptionally includes a pharmaceutically acceptable carrier and/or anencapsulating agent, such as a lipid or polymer micro- or nano-particle,micelle, liposome, etc. In other embodiments, a genome editing system isimplemented as one or more nucleic acids encoding the RNA-guidednuclease and gRNA components described above (optionally with one ormore additional components); in still other embodiments, the genomeediting system is implemented as one or more vectors comprising suchnucleic acids, for example a viral vector such as an AAV; and in stillother embodiments, the genome editing system is implemented as acombination of any of the foregoing. Additional or modifiedimplementations that operate according to the principles set forthherein will be apparent to the skilled artisan and are within the scopeof this disclosure.

It should be noted that the genome editing systems of the presentinvention can be targeted to a single specific nucleotide sequence, orcan be targeted to—and capable of editing in parallel—two or morespecific nucleotide sequences through the use of two or more gRNAs. Theuse of two or more gRNAs targeted to different sites is referred to as“multiplexing” throughout this disclosure, and can be employed to targetmultiple, unrelated target sequences of interest, or to form multipleSSBs and/or DSBs within a single target domain and, in some cases, togenerate specific edits within such target domain. For example, thisdisclosure and International Patent Publication No. WO2015/138510 byMaeder et al. (“Maeder”), which is incorporated by reference herein,both describe a genome editing system for correcting a point mutation(C.2991+1655A to G) in the human CEP290 gene that results in thecreation of a cryptic splice site, which in turn reduces or eliminatesthe function of the gene. The genome editing system of Maeder utilizestwo gRNAs targeted to sequences on either side of (i.e., flanking) thepoint mutation, and forms DSBs that flank the mutation. This, in turn,promotes deletion of the intervening sequence, including the mutation,thereby eliminating the cryptic splice site and restoring normal genefunction.

As another example, International Patent Publication No. WO2016/073990by Cotta-Ramusino et al. (“Cotta-Ramusino”), incorporated by referenceherein, describes a genome editing system that utilizes two gRNAs incombination with a Cas9 nickase (a Cas9 that makes a single strand nicksuch as S. pyogenes D10A), an arrangement termed a “dual-nickasesystem.” The dual-nickase system of Cotta-Ramusino is configured to maketwo nicks on opposite strands of a sequence of interest that are offsetby one or more nucleotides, which nicks combine to create a doublestrand break having an overhang (5′ in the case of Cotta-Ramusino,though 3′ overhangs are also possible). The overhang, in turn, canfacilitate homology directed repair events in some circumstances. Asanother example, International Patent Publication No. WO2015/070083 byZhang et al., incorporated by reference herein, describes a gRNAtargeted to a nucleotide sequence encoding Cas9 (referred to as a“governing” gRNA), which can be included in a genome editing systemcomprising one or more additional gRNAs to permit transient expressionof a Cas9 that might otherwise be constitutively expressed, for examplein some virally transduced cells. These multiplexing applications areintended to be exemplary, rather than limiting, and the skilled artisanwill appreciate that other applications of multiplexing are generallycompatible with the genome editing systems described here.

Genome editing systems can, in some instances, form double strand breaksthat are repaired by cellular DNA double-strand break mechanisms such asnon-homologous end joining (NHEJ), or homology directed repair (HDR).These mechanisms are described throughout the literature (see, e.g.,Davis 2014 (describing Alt-HDR), Frit 2014 (describing Alt-NHEJ), andIyama 2013 (describing canonical HDR and NHEJ pathways generally), allof which are incorporated by reference herein).

Where genome editing systems operate by forming DSBs, such systemsoptionally include one or more components that promote or facilitate aparticular mode of double-strand break repair or a particular repairoutcome. For example, Cotta-Ramusino also describes genome editingsystems in which a single stranded oligonucleotide “donor template” isadded; the donor template is incorporated into a target region ofcellular DNA that is cleaved by the genome editing system, and canresult in a change in the target sequence.

In other cases, genome editing systems modify a target sequence, ormodify expression of a gene in or near the target sequence, withoutcausing single- or double-strand breaks. For example, a genome editingsystem can include an RNA-guided nuclease/cytidine deaminase fusionprotein, and can operate by generating targeted C-to-A substitutions.Suitable nuclease/deaminase fusions are described in Komor 2016, whichis incorporated by reference. Alternatively, a genome editing system canutilize a cleavage-inactivated (i.e., a “dead”) nuclease, such as a deadCas9, and can operate by forming stable complexes on one or moretargeted regions of cellular DNA, thereby interfering with functionsinvolving the targeted region(s) such as mRNA transcription andchromatin remodeling.

II. gRNA Molecules

The terms guide RNA and gRNA refer to any nucleic acid that promotes thespecific association (or “targeting”) of an RNA-guided nuclease such asa Cas9 or a Cpf1 to a target sequence such as a genomic or episomalsequence in a cell. gRNAs can be unimolecular (comprising a single RNAmolecule, and referred to alternatively as chimeric), or modular(comprising more than one, and typically two, separate RNA molecules,such as a crRNA and a tracrRNA, which are usually associated with oneanother, for example by duplexing). gRNAs and their component parts aredescribed throughout the literature (see, e.g., Briner 2014, which isincorporated by reference; see also Cotta-Ramusino).

In bacteria and archea, type II CRISPR systems generally comprise anRNA-guided nuclease protein such as Cas9, a CRISPR RNA (crRNA) thatincludes a 5′ region that is complementary to a foreign sequence, and atrans-activating crRNA (tracrRNA) that includes a 5′ region that iscomplementary to, and forms a duplex with, a 3′ region of the crRNA.While not intending to be bound by any theory, it is thought that thisduplex facilitates the formation of—and is necessary for the activityof—the Cas9/gRNA complex. As type II CRISPR systems were adapted for usein gene editing, it was discovered that the crRNA and tracrRNA could bejoined into a single unimolecular or chimeric gRNA, for example by meansof a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequencebridging complementary regions of the crRNA (at its 3′ end) and thetracrRNA (at its 5′ end) (Mali 2013; Jiang 2013; Jinek 2012; allincorporated by reference herein).

gRNAs, whether unimolecular or modular, include a targeting domain thatis fully or partially complementary to a target domain within a targetsequence, such as a DNA sequence in the genome of a cell where editingis desired. In certain embodiments, this target sequence encompasses oris proximal to a CEP290 target position. Targeting domains are referredto by various names in the literature, including without limitation“guide sequences” (Hsu 2013, incorporated by reference herein),“complementarity regions” (Cotta-Ramusino), “spacers” (Briner 2014), andgenerically as “crRNAs” (Jiang 2013). Irrespective of the names they aregiven, targeting domains are typically 10-30 nucleotides in length,preferably 16-24 nucleotides in length (for example, 16, 17, 18, 19, 20,21, 22, 23 or 24 nucleotides in length), and are at or near the 5′terminus of in the case of a Cas9 gRNA, and at or near the 3′ terminusin the case of a Cpf1 gRNA.

In addition to the targeting domains, gRNAs typically (but notnecessarily, as discussed below) include a plurality of domains thatinfluence the formation or activity of gRNA/Cas9 complexes. For example,as mentioned above, the duplexed structure formed by first and secondarycomplementarity domains of a gRNA (also referred to as arepeat:anti-repeat duplex) interacts with the recognition (REC) lobe ofCas9 and may mediate the formation of Cas9/gRNA complexes (Nishimasu2014; Nishimasu 2015; both incorporated by reference herein). It shouldbe noted that the first and/or second complementarity domains cancontain one or more poly-A tracts, which can be recognized by RNApolymerases as a termination signal. The sequence of the first andsecond complementarity domains are, therefore, optionally modified toeliminate these tracts and promote the complete in vitro transcriptionof gRNAs, for example through the use of A-G swaps as described inBriner 2014, or A-U swaps. These and other similar modifications to thefirst and second complementarity domains are within the scope of thepresent disclosure.

Along with the first and second complementarity domains, Cas9 gRNAstypically include two or more additional duplexed regions that arenecessary for nuclease activity in vivo but not necessarily in vitro(Nishimasu 2015). A first stem-loop near the 3′ portion of the secondcomplementarity domain is referred to variously as the “proximal domain”(Cotta-Ramusino) “stem loop 1” (Nishimasu 2014; Nishimasu 2015) and the“nexus” (Briner 2014). One or more additional stem loop structures aregenerally present near the 3′ end of the gRNA, with the number varyingby species: S. pyogenes gRNAs typically include two 3′ stem loops (for atotal of four stem loop structures including the repeat:anti-repeatduplex), while s. aureus and other species have only one (for a total ofthree). A description of conserved stem loop structures (and gRNAstructures more generally) organized by species is provided in Briner2014.

Skilled artisans will appreciate that gRNAs can be modified in a numberof ways, some of which are described below, and these modifications arewithin the scope of disclosure. For economy of presentation in thisdisclosure, gRNAs may be presented by reference solely to theirtargeting domain sequences.

A gRNA molecule comprises a number of domains. The gRNA molecule domainsare described in more detail below.

Several exemplary gRNA structures, with domains indicated thereon, areprovided in FIG. 1. While not wishing to be bound by theory, with regardto the three dimensional form, or intra- or inter-strand interactions ofan active form of a gRNA, regions of high complementarity are sometimesshown as duplexes in FIG. 1 and other depictions provided herein.

In an embodiment, a unimolecular, or chimeric, gRNA comprises,preferably from 5′ to 3′:

-   -   a targeting domain (which is complementary to a target nucleic        acid in the CEP290 gene, e.g., a targeting domain from any of        Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables        6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B,        or Table 11);    -   a first complementarity domain;    -   a linking domain;    -   a second complementarity domain (which is complementary to the        first complementarity domain);    -   a proximal domain; and    -   optionally, a tail domain.

In an embodiment, a modular gRNA comprises:

-   -   a first strand comprising, preferably from 5′ to 3′;        -   a targeting domain (which is complementary to a target            nucleic acid in the CEP290 gene, e.g., a targeting domain            from Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D,            Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E,            Tables 10A-10B, or Table 11); and        -   a first complementarity domain; and    -   a second strand, comprising, preferably from 5′ to 3′:        -   optionally, a 5′ extension domain;        -   a second complementarity domain;        -   a proximal domain; and        -   optionally, a tail domain.

The domains are discussed briefly below.

Targeting Domain

FIGS. 1A-1G provide examples of the placement of targeting domains.

The targeting domain comprises a nucleotide sequence that iscomplementary, e.g., at least 80, 85, 90, or 95% complementary, e.g.,fully complementary, to the target sequence on the target nucleic acid.The targeting domain is part of an RNA molecule and will thereforecomprise the base uracil (U), while any DNA encoding the gRNA moleculewill comprise the base thymine (T). While not wishing to be bound bytheory, in an embodiment, it is believed that the complementarity of thetargeting domain with the target sequence contributes to specificity ofthe interaction of the gRNA molecule/Cas9 molecule complex with a targetnucleic acid. It is understood that in a targeting domain and targetsequence pair, the uracil bases in the targeting domain will pair withthe adenine bases in the target sequence. In an embodiment, the targetdomain itself comprises in the 5′ to 3′ direction, an optional secondarydomain, and a core domain. In an embodiment, the core domain is fullycomplementary with the target sequence.

In an embodiment, the targeting domain is 5 to 50 nucleotides in length.The strand of the target nucleic acid with which the targeting domain iscomplementary is referred to herein as the complementary strand. Some orall of the nucleotides of the domain can have a modification, e.g., amodification found in Section VIII herein.

In an embodiment, the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain is 25 nucleotides in length.

In an embodiment, the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises 16 nucleotides.

In an embodiment, the targeting domain comprises 17 nucleotides.

In an embodiment, the targeting domain comprises 18 nucleotides.

In an embodiment, the targeting domain comprises 19 nucleotides.

In an embodiment, the targeting domain comprises 20 nucleotides.

In an embodiment, the targeting domain comprises 21 nucleotides.

In an embodiment, the targeting domain comprises 22 nucleotides.

In an embodiment, the targeting domain comprises 23 nucleotides.

In an embodiment, the targeting domain comprises 24 nucleotides.

In an embodiment, the targeting domain comprises 25 nucleotides.

In an embodiment, the targeting domain comprises 26 nucleotides.

Targeting domains are discussed in more detail below.

First Complementarity Domain

FIGS. 1A-1G provide examples of first complementarity domains.

The first complementarity domain is complementary with the secondcomplementarity domain, and in an embodiment, has sufficientcomplementarity to the second complementarity domain to form a duplexedregion under at least some physiological conditions. In an embodiment,the first complementarity domain is 5 to 30 nucleotides in length. In anembodiment, the first complementarity domain is 5 to 25 nucleotides inlength. In an embodiment, the first complementary domain is 7 to 25nucleotides in length. In an embodiment, the first complementary domainis 7 to 22 nucleotides in length. In an embodiment, the firstcomplementary domain is 7 to 18 nucleotides in length. In an embodiment,the first complementary domain is 7 to 15 nucleotides in length. In anembodiment, the first complementary domain is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength.

In an embodiment, the first complementarity domain comprises 3subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, acentral subdomain, and a 3′ subdomain. In an embodiment, the 5′subdomain is 4-9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In anembodiment, the central subdomain is 1, 2, or 3, e.g., 1, nucleotide inlength. In an embodiment, the 3′ subdomain is 3 to 25, e.g., 4-22, 4-18,or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25, nucleotides in length.

The first complementarity domain can share homology with, or be derivedfrom, a naturally occurring first complementarity domain. In anembodiment, it has at least 50% homology with a first complementaritydomain disclosed herein, e.g., an S. pyogenes, S. aureus, or S.thermophilus, first complementarity domain.

Some or all of the nucleotides of the domain can have a modification,e.g., a modification found in Section VIII herein.

First complementarity domains are discussed in more detail below.

Linking Domain

FIGS. 1A-1G provide examples of linking domains.

A linking domain serves to link the first complementarity domain withthe second complementarity domain of a unimolecular gRNA. The linkingdomain can link the first and second complementarity domains covalentlyor non-covalently. In an embodiment, the linkage is covalent. In anembodiment, the linking domain covalently couples the first and secondcomplementarity domains, see, e.g., FIGS. 1B-1E. In an embodiment, thelinking domain is, or comprises, a covalent bond interposed between thefirst complementarity domain and the second complementarity domain.Typically the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6,7, 8, 9, or 10 nucleotides.

In modular gRNA molecules the two molecules are associated by virtue ofthe hybridization of the complementarity domains see e.g., FIG. 1A.

A wide variety of linking domains are suitable for use in unimoleculargRNA molecules. Linking domains can consist of a covalent bond, or be asshort as one or a few nucleotides, e.g., 1, 2, 3, 4, or 5 nucleotides inlength. In an embodiment, a linking domain is 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 or more nucleotides in length. In an embodiment, alinking domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5nucleotides in length. In an embodiment, a linking domain shareshomology with, or is derived from, a naturally occurring sequence, e.g.,the sequence of a tracrRNA that is 5′ to the second complementaritydomain. In an embodiment, the linking domain has at least 50% homologywith a linking domain disclosed herein.

Some or all of the nucleotides of the domain can have a modification,e.g., a modification found in Section VIII herein.

Linking domains are discussed in more detail below.

5′ Extension Domain

In an embodiment, a modular gRNA can comprise additional sequence, 5′ tothe second complementarity domain, referred to herein as the 5′extension domain, see, e.g., FIG. 1A. In an embodiment, the 5′ extensiondomain is, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4 nucleotides in length.In an embodiment, the 5′ extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or10 or more nucleotides in length.

Second Complementarity Domain

FIGS. 1A-1G provide examples of second complementarity domains.

The second complementarity domain is complementary with the firstcomplementarity domain, and in an embodiment, has sufficientcomplementarity to the second complementarity domain to form a duplexedregion under at least some physiological conditions. In an embodiment,e.g., as shown in FIGS. 1A-1B, the second complementarity domain caninclude sequence that lacks complementarity with the firstcomplementarity domain, e.g., sequence that loops out from the duplexedregion.

In an embodiment, the second complementarity domain is 5 to 27nucleotides in length. In an embodiment, it is longer than the firstcomplementarity region. In an embodiment the second complementary domainis 7 to 27 nucleotides in length. In an embodiment, the secondcomplementary domain is 7 to 25 nucleotides in length. In an embodiment,the second complementary domain is 7 to 20 nucleotides in length. In anembodiment, the second complementary domain is 7 to 17 nucleotides inlength. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26nucleotides in length.

In an embodiment, the second complementarity domain comprises 3subdomains, which, in the 5′ to 3′ direction are: a 5′ subdomain, acentral subdomain, and a 3′ subdomain. In an embodiment, the 5′subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25 nucleotides in length. In an embodiment, the central subdomain is 1,2, 3, 4 or 5, e.g., 3, nucleotides in length. In an embodiment, the 3′subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.

In an embodiment, the 5′ subdomain and the 3′ subdomain of the firstcomplementarity domain, are respectively, complementary, e.g., fullycomplementary, with the 3′ subdomain and the 5′ subdomain of the secondcomplementarity domain.

The second complementarity domain can share homology with or be derivedfrom a naturally occurring second complementarity domain. In anembodiment, it has at least 50% homology with a second complementaritydomain disclosed herein, e.g., an S. pyogenes, S. aureus, or S.thermophilus, first complementarity domain.

Some or all of the nucleotides of the domain can have a modification,e.g., a modification found in Section VIII herein.

Proximal Domain

FIGS. 1A-1G provide examples of proximal domains.

In an embodiment, the proximal domain is 5 to 20 nucleotides in length.In an embodiment, the proximal domain can share homology with or bederived from a naturally occurring proximal domain. In an embodiment, ithas at least 50% homology with a proximal domain disclosed herein, e.g.,an S. pyogenes, S. aureus, or S. thermophilus, proximal domain.

Some or all of the nucleotides of the domain can have a modification,e.g., a modification found in Section VIII herein.

Tail Domain

FIGS. 1A-1G provide examples of tail domains.

As can be seen by inspection of the tail domains in FIGS. 1A and 1B-1F,a broad spectrum of tail domains are suitable for use in gRNA molecules.In an embodiment, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotides in length. In embodiment, the tail domainnucleotides are from or share homology with sequence from the 5′ end ofa naturally occurring tail domain, see e.g., FIG. 1D or 1E. In anembodiment, the tail domain includes sequences that are complementary toeach other and which, under at least some physiological conditions, forma duplexed region.

In an embodiment, the tail domain is absent or is 1 to 50 nucleotides inlength. In an embodiment, the tail domain can share homology with or bederived from a naturally occurring proximal tail domain. In anembodiment, it has at least 50% homology with a tail domain disclosedherein, e.g., an S. pyogenes, S. aureus, or S. thermophilus, taildomain.

In an embodiment, the tail domain includes nucleotides at the 3′ endthat are related to the method of in vitro or in vivo transcription.When a T7 promoter is used for in vitro transcription of the gRNA, thesenucleotides may be any nucleotides present before the 3′ end of the DNAtemplate. When a U6 promoter is used for in vivo transcription, thesenucleotides may be the sequence UUUUUU. When alternate pol-III promotersare used, these nucleotides may be various numbers or uracil bases ormay include alternate bases.

The domains of gRNA molecules are described in more detail below.

Targeting Domain

The “targeting domain” of the gRNA is complementary to the “targetdomain” on the target nucleic acid. The strand of the target nucleicacid comprising the core domain target is referred to herein as the“complementary strand” of the target nucleic acid. Guidance on theselection of targeting domains can be found, e.g., in Fu 2014 andSternberg 2014.

In an embodiment, the targeting domain is 16, 17, 18, 19, 20, 21, 22,23, 24, 25 or 26 nucleotides in length. In the figures and sequencelisting provided herein, targeting domains are generally shown with 20nucleotides. In each of these instances, the targeting domain mayactually be shorter or longer as disclosed herein, for example from 16to 26 nucleotides.

In an embodiment, the targeting domain is 16 nucleotides in length.

In an embodiment, the targeting domain is 17 nucleotides in length.

In an embodiment, the targeting domain is 18 nucleotides in length.

In an embodiment, the targeting domain is 19 nucleotides in length.

In an embodiment, the targeting domain is 20 nucleotides in length.

In an embodiment, the targeting domain is 21 nucleotides in length.

In an embodiment, the targeting domain is 22 nucleotides in length.

In an embodiment, the targeting domain is 23 nucleotides in length.

In an embodiment, the targeting domain is 24 nucleotides in length.

In an embodiment, the targeting domain is 25 nucleotides in length.

In an embodiment, the targeting domain is 26 nucleotides in length.

In an embodiment, the targeting domain comprises 16 nucleotides.

In an embodiment, the targeting domain comprises 17 nucleotides.

In an embodiment, the targeting domain comprises 18 nucleotides.

In an embodiment, the targeting domain comprises 19 nucleotides.

In an embodiment, the targeting domain comprises 20 nucleotides.

In an embodiment, the targeting domain comprises 21 nucleotides.

In an embodiment, the targeting domain comprises 22 nucleotides.

In an embodiment, the targeting domain comprises 23 nucleotides.

In an embodiment, the targeting domain comprises 24 nucleotides.

In an embodiment, the targeting domain comprises 25 nucleotides.

In an embodiment, the targeting domain comprises 26 nucleotides.

In an embodiment, the targeting domain is 10+/−5, 20+/−5, 30+/−5,40+/−5, 50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides,in length.

In an embodiment, the targeting domain is 20+/−5 nucleotides in length.

In an embodiment, the targeting domain is 20+/−10, 30+/−10, 40+/−10,50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, inlength.

In an embodiment, the targeting domain is 30+/−10 nucleotides in length.

In an embodiment, the targeting domain is 10 to 100, 10 to 90, 10 to 80,10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15nucleotides in length. In other embodiments, the targeting domain is 20to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20to 30, or 20 to 25 nucleotides in length.

Typically the targeting domain has full complementarity with the targetsequence. In some embodiments the targeting domain has or includes 1, 2,3, 4, 5, 6, 7 or 8 nucleotides that are not complementary with thecorresponding nucleotide of the targeting domain.

In an embodiment, the target domain includes 1, 2, 3, 4 or 5 nucleotidesthat are complementary with the corresponding nucleotide of thetargeting domain within 5 nucleotides of its 5′ end. In an embodiment,the target domain includes 1, 2, 3, 4 or 5 nucleotides that arecomplementary with the corresponding nucleotide of the targeting domainwithin 5 nucleotides of its 3′ end.

In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotidesthat are not complementary with the corresponding nucleotide of thetargeting domain within 5 nucleotides of its 5′ end. In an embodiment,the target domain includes 1, 2, 3, or 4 nucleotides that are notcomplementary with the corresponding nucleotide of the targeting domainwithin 5 nucleotides of its 3′ end.

In an embodiment, the degree of complementarity, together with otherproperties of the gRNA, is sufficient to allow targeting of a Cas9molecule to the target nucleic acid.

In some embodiments, the targeting domain comprises two consecutivenucleotides that are not complementary to the target domain(“non-complementary nucleotides”), e.g., two consecutivenoncomplementary nucleotides that are within 5 nucleotides of the 5′ endof the targeting domain, within 5 nucleotides of the 3′ end of thetargeting domain, or more than 5 nucleotides away from one or both endsof the targeting domain.

In an embodiment, no two consecutive nucleotides within 5 nucleotides ofthe 5′ end of the targeting domain, within 5 nucleotides of the 3′ endof the targeting domain, or within a region that is more than 5nucleotides away from one or both ends of the targeting domain, are notcomplementary to the targeting domain.

In an embodiment, there are no noncomplementary nucleotides within 5nucleotides of the 5′ end of the targeting domain, within 5 nucleotidesof the 3′ end of the targeting domain, or within a region that is morethan 5 nucleotides away from one or both ends of the targeting domain.

In an embodiment, the targeting domain nucleotides do not comprisemodifications, e.g., modifications of the type provided in Section VIII.However, in an embodiment, the targeting domain comprises one or moremodifications, e.g., modifications that it render it less susceptible todegradation or more bio-compatible, e.g., less immunogenic. By way ofexample, the backbone of the targeting domain can be modified with aphosphorothioate, or other modification(s) from Section VIII. In anembodiment, a nucleotide of the targeting domain can comprise a 2′modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or othermodification(s) from Section VIII.

In some embodiments, the targeting domain includes 1, 2, 3, 4, 5, 6, 7or 8 or more modifications. In an embodiment, the targeting domainincludes 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′ end.In an embodiment, the targeting domain comprises as many as 1, 2, 3, or4 modifications within 5 nucleotides of its 3′ end.

In some embodiments, the targeting domain comprises modifications at twoconsecutive nucleotides, e.g., two consecutive nucleotides that arewithin 5 nucleotides of the 5′ end of the targeting domain, within 5nucleotides of the 3′ end of the targeting domain, or more than 5nucleotides away from one or both ends of the targeting domain.

In an embodiment, no two consecutive nucleotides are modified within 5nucleotides of the 5′ end of the targeting domain, within 5 nucleotidesof the 3′ end of the targeting domain, or within a region that is morethan 5 nucleotides away from one or both ends of the targeting domain.In an embodiment, no nucleotide is modified within 5 nucleotides of the5′ end of the targeting domain, within 5 nucleotides of the 3′ end ofthe targeting domain, or within a region that is more than 5 nucleotidesaway from one or both ends of the targeting domain.

Modifications in the targeting domain can be selected to not interferewith targeting efficacy, which can be evaluated by testing a candidatemodification in the system described in Section V. gRNAs having acandidate targeting domain having a selected length, sequence, degree ofcomplementarity, or degree of modification, can be evaluated in a systemin Section V. The candidate targeting domain can be placed, eitheralone, or with one or more other candidate changes in a gRNAmolecule/Cas9 molecule system known to be functional with a selectedtarget and evaluated.

In some embodiments, all of the modified nucleotides are complementaryto and capable of hybridizing to corresponding nucleotides present inthe target domain. In other embodiments, 1, 2, 3, 4, 5, 6, 7 or 8 ormore modified nucleotides are not complementary to or capable ofhybridizing to corresponding nucleotides present in the target domain.

In an embodiment, the targeting domain comprises, preferably in the5′→3′ direction: a secondary domain and a core domain. These domains arediscussed in more detail below.

Core Domain and Secondary Domain of the Targeting Domain

The “core domain” of the targeting domain is complementary to the “coredomain target” on the target nucleic acid. In an embodiment, the coredomain comprises about 8 to about 13 nucleotides from the 3′ end of thetargeting domain (e.g., the most 3′ 8 to 13 nucleotides of the targetingdomain).

In an embodiment, the secondary domain is absent or optional.

In an embodiment, the core domain and targeting domain, areindependently, 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2,13+/−2, 14+/−2, 15+/−2, 16+−2, 17+/−2, or 18+/−2, nucleotides in length.

In an embodiment, the core domain and targeting domain, areindependently, 10+/−2 nucleotides in length.

In an embodiment, the core domain and targeting domain, areindependently, 10+/−4 nucleotides in length.

In an embodiment, the core domain and targeting domain, areindependently, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18,nucleotides in length.

In an embodiment, the core domain and targeting domain, areindependently 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to20 10 to 20 or 15 to 20 nucleotides in length.

In an embodiment, the core domain and targeting domain, areindependently 3 to 15, e.g., 6 to 15, 7 to 14, 7 to 13, 6 to 12, 7 to12, 7 to 11, 7 to 10, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10 or 8to 9 nucleotides in length.

The core domain is complementary with the core domain target. Typicallythe core domain has exact complementarity with the core domain target.In some embodiments, the core domain can have 1, 2, 3, 4 or 5nucleotides that are not complementary with the corresponding nucleotideof the core domain. In an embodiment, the degree of complementarity,together with other properties of the gRNA, is sufficient to allowtargeting of a Cas9 molecule to the target nucleic acid.

The “secondary domain” of the targeting domain of the gRNA iscomplementary to the “secondary domain target” of the target nucleicacid.

In an embodiment, the secondary domain is positioned 5′ to the coredomain.

In an embodiment, the secondary domain is absent or optional.

In an embodiment, if the targeting domain is 26 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 12 to 17nucleotides in length.

In an embodiment, if the targeting domain is 25 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 12 to 17nucleotides in length.

In an embodiment, if the targeting domain is 24 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 11 to 16nucleotides in length.

In an embodiment, if the targeting domain is 23 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 10 to 15nucleotides in length.

In an embodiment, if the targeting domain is 22 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 9 to 14nucleotides in length.

In an embodiment, if the targeting domain is 21 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 8 to 13nucleotides in length.

In an embodiment, if the targeting domain is 20 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 7 to 12nucleotides in length.

In an embodiment, if the targeting domain is 19 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 6 to 11nucleotides in length.

In an embodiment, if the targeting domain is 18 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 5 to 10nucleotides in length.

In an embodiment, if the targeting domain is 17 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 4 to 9nucleotides in length.

In an embodiment, if the targeting domain is 16 nucleotides in lengthand the core domain (counted from the 3′ end of the targeting domain) is8 to 13 nucleotides in length, the secondary domain is 3 to 8nucleotides in length.

In an embodiment, the secondary domain is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15 nucleotides in length.

The secondary domain is complementary with the secondary domain target.Typically the secondary domain has exact complementarity with thesecondary domain target. In some embodiments the secondary domain canhave 1, 2, 3, 4 or 5 nucleotides that are not complementary with thecorresponding nucleotide of the secondary domain. In an embodiment, thedegree of complementarity, together with other properties of the gRNA,is sufficient to allow targeting of a Cas9 molecule to the targetnucleic acid.

In an embodiment, the core domain nucleotides do not comprisemodifications, e.g., modifications of the type provided in Section VIII.However, in an embodiment, the core domain comprise one or moremodifications, e.g., modifications that it render it less susceptible todegradation or more bio-compatible, e.g., less immunogenic. By way ofexample, the backbone of the core domain can be modified with aphosphorothioate, or other modification(s) from Section VIII. In anembodiment a nucleotide of the core domain can comprise a 2′modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or othermodification(s) from Section VIII. Typically, a core domain will containno more than 1, 2, or 3 modifications.

Modifications in the core domain can be selected to not interfere withtargeting efficacy, which can be evaluated by testing a candidatemodification in the system described in Section V. gRNA's having acandidate core domain having a selected length, sequence, degree ofcomplementarity, or degree of modification, can be evaluated in thesystem described at Section V. The candidate core domain can be placed,either alone, or with one or more other candidate changes in a gRNAmolecule/Cas9 molecule system known to be functional with a selectedtarget and evaluated.

In an embodiment, the secondary domain nucleotides do not comprisemodifications, e.g., modifications of the type provided in Section VIII.However, in an embodiment, the secondary domain comprises one or moremodifications, e.g., modifications that it render it less susceptible todegradation or more bio-compatible, e.g., less immunogenic. By way ofexample, the backbone of the secondary domain can be modified with aphosphorothioate, or other modification(s) from Section VIII. In anembodiment a nucleotide of the secondary domain can comprise a 2′modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or othermodification(s) from Section VIII. Typically, a secondary domain willcontain no more than 1, 2, or 3 modifications.

Modifications in the secondary domain can be selected to not interferewith targeting efficacy, which can be evaluated by testing a candidatemodification in the system described in Section V. gRNA's having acandidate secondary domain having a selected length, sequence, degree ofcomplementarity, or degree of modification, can be evaluated in thesystem described at Section V. The candidate secondary domain can beplaced, either alone, or with one or more other candidate changes in agRNA molecule/Cas9 molecule system known to be functional with aselected target and evaluated.

In an embodiment, (1) the degree of complementarity between the coredomain and its target, and (2) the degree of complementarity between thesecondary domain and its target, may differ. In an embodiment, (1) maybe greater (2). In an embodiment, (1) may be less than (2). In anembodiment, (1) and (2) are the same, e.g., each may be completelycomplementary with its target.

In an embodiment, (1) the number of modification (e.g., modificationsfrom Section VIII) of the nucleotides of the core domain and (2) thenumber of modification (e.g., modifications from Section VIII) of thenucleotides of the secondary domain, may differ. In an embodiment, (1)may be less than (2). In an embodiment, (1) may be greater than (2). Inan embodiment, (1) and (2) may be the same, e.g., each may be free ofmodifications.

First and Second Complementarity Domains

The first complementarity domain is complementary with the secondcomplementarity domain.

Typically the first domain does not have exact complementarity with thesecond complementarity domain target. In some embodiments, the firstcomplementarity domain can have 1, 2, 3, 4 or 5 nucleotides that are notcomplementary with the corresponding nucleotide of the secondcomplementarity domain. In an embodiment, 1, 2, 3, 4, 5 or 6, e.g., 3nucleotides, will not pair in the duplex, and, e.g., form a non-duplexedor looped-out region. In an embodiment, an unpaired, or loop-out,region, e.g., a loop-out of 3 nucleotides, is present on the secondcomplementarity domain. In an embodiment, the unpaired region begins 1,2, 3, 4, 5, or 6, e.g., 4, nucleotides from the 5′ end of the secondcomplementarity domain.

In an embodiment, the degree of complementarity, together with otherproperties of the gRNA, is sufficient to allow targeting of a Cas9molecule to the target nucleic acid.

In an embodiment, the first and second complementarity domains are:

independently, 6+/−2, 7+/−2, 8+/−2, 9+/−2, 10+/−2, 11+/−2, 12+/−2,13+/−2, 14+/−2, 15+/−2, 16+/−2, 17+/−2, 18+/−2, 19+/−2, or 20+/−2,21+/−2, 22+/−2, 23+/−2, or 24+/−2 nucleotides in length;

independently, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, or 26, nucleotides in length; or

independently, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 7 to 18, 9to 16, or 10 to 14 nucleotides in length.

In an embodiment, the second complementarity domain is longer than thefirst complementarity domain, e.g., 2, 3, 4, 5, or 6, e.g., 6,nucleotides longer.

In an embodiment, the first and second complementary domains,independently, do not comprise modifications, e.g., modifications of thetype provided in Section VIII.

In an embodiment, the first and second complementary domains,independently, comprise one or more modifications, e.g., modificationsthat the render the domain less susceptible to degradation or morebio-compatible, e.g., less immunogenic. By way of example, the backboneof the domain can be modified with a phosphorothioate, or othermodification(s) from Section VIII. In an embodiment a nucleotide of thedomain can comprise a 2′ modification, e.g., a 2-acetylation, e.g., a 2′methylation, or other modification(s) from Section VIII.

In an embodiment, the first and second complementary domains,independently, include 1, 2, 3, 4, 5, 6, 7 or 8 or more modifications.In an embodiment, the first and second complementary domains,independently, include 1, 2, 3, or 4 modifications within 5 nucleotidesof its 5′ end. In an embodiment, the first and second complementarydomains, independently, include as many as 1, 2, 3, or 4 modificationswithin 5 nucleotides of its 3′ end.

In an embodiment, the first and second complementary domains,independently, include modifications at two consecutive nucleotides,e.g., two consecutive nucleotides that are within 5 nucleotides of the5′ end of the domain, within 5 nucleotides of the 3′ end of the domain,or more than 5 nucleotides away from one or both ends of the domain. Inan embodiment, the first and second complementary domains,independently, include no two consecutive nucleotides that are modified,within 5 nucleotides of the 5′ end of the domain, within 5 nucleotidesof the 3′ end of the domain, or within a region that is more than 5nucleotides away from one or both ends of the domain. In an embodiment,the first and second complementary domains, independently, include nonucleotide that is modified within 5 nucleotides of the 5′ end of thedomain, within 5 nucleotides of the 3′ end of the domain, or within aregion that is more than 5 nucleotides away from one or both ends of thedomain.

Modifications in a complementarity domain can be selected to notinterfere with targeting efficacy, which can be evaluated by testing acandidate modification in the system described in Section V. gRNA'shaving a candidate complementarity domain having a selected length,sequence, degree of complementarity, or degree of modification, can beevaluated in the system described in Section V. The candidatecomplementarity domain can be placed, either alone, or with one or moreother candidate changes in a gRNA molecule/Cas9 molecule system known tobe functional with a selected target and evaluated.

In an embodiment, the first complementarity domain has at least 60, 70,80, 85%, 90% or 95% homology with, or differs by no more than 1, 2, 3,4, 5, or 6 nucleotides from, a reference first complementarity domain,e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus, or S.thermophilus, first complementarity domain, or a first complementaritydomain described herein, e.g., from FIGS. 1A-1G.

In an embodiment, the second complementarity domain has at least 60, 70,80, 85%, 90%, or 95% homology with, or differs by no more than 1, 2, 3,4, 5, or 6 nucleotides from, a reference second complementarity domain,e.g., a naturally occurring, e.g., an S. pyogenes, S. aureus, or S.thermophilus, second complementarity domain, or a second complementaritydomain described herein, e.g., from FIG. 1A-1G.

The duplexed region formed by first and second complementarity domainsis typically 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21or 22 base pairs in length (excluding any looped out or unpairednucleotides).

In some embodiments, the first and second complementarity domains, whenduplexed, comprise 11 paired nucleotides, for example, in the gRNAsequence (one paired strand underlined, one bolded):

(SEQ ID NO: 5) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC.

In some embodiments, the first and second complementarity domains, whenduplexed, comprise 15 paired nucleotides, for example in the gRNAsequence (one paired strand underlined, one bolded):

(SEQ ID NO: 27) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAG UCGGUGC.

In some embodiments the first and second complementarity domains, whenduplexed, comprise 16 paired nucleotides, for example in the gRNAsequence (one paired strand underlined, one bolded):

(SEQ ID NO: 28) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGGAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGC.

In some embodiments the first and second complementarity domains, whenduplexed, comprise 21 paired nucleotides, for example in the gRNAsequence (one paired strand underlined, one bolded):

(SEQ ID NO: 29) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUGGAAACAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC.

In some embodiments, nucleotides are exchanged to remove poly-U tracts,for example in the gRNA sequences (exchanged nucleotides underlined):

(SEQ ID NO: 30) NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAGAAAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; (SEQ ID NO: 31)NNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC; and (SEQ ID NO: 32)NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAUGCUGUAUUGGAAACAAUACAGCAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC.5′ Extension Domain

In an embodiment, a modular gRNA can comprise additional sequence, 5′ tothe second complementarity domain. In an embodiment, the 5′ extensiondomain is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4nucleotides in length. In an embodiment, the 5′ extension domain is 2,3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.

In an embodiment, the 5′ extension domain nucleotides do not comprisemodifications, e.g., modifications of the type provided in Section VIII.However, in an embodiment, the 5′ extension domain comprises one or moremodifications, e.g., modifications that it render it less susceptible todegradation or more bio-compatible, e.g., less immunogenic. By way ofexample, the backbone of the 5′ extension domain can be modified with aphosphorothioate, or other modification(s) from Section VIII. In anembodiment, a nucleotide of the 5′ extension domain can comprise a 2′modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or othermodification(s) from Section VIII.

In some embodiments, the 5′ extension domain can comprise as many as 1,2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the 5′ extensiondomain comprises as many as 1, 2, 3, or 4 modifications within 5nucleotides of its 5′ end, e.g., in a modular gRNA molecule. In anembodiment, the 5′ extension domain comprises as many as 1, 2, 3, or 4modifications within 5 nucleotides of its 3′ end, e.g., in a modulargRNA molecule.

In some embodiments, the 5′ extension domain comprises modifications attwo consecutive nucleotides, e.g., two consecutive nucleotides that arewithin 5 nucleotides of the 5′ end of the 5′ extension domain, within 5nucleotides of the 3′ end of the 5′ extension domain, or more than 5nucleotides away from one or both ends of the 5′ extension domain. In anembodiment, no two consecutive nucleotides are modified within 5nucleotides of the 5′ end of the 5′ extension domain, within 5nucleotides of the 3′ end of the 5′ extension domain, or within a regionthat is more than 5 nucleotides away from one or both ends of the 5′extension domain. In an embodiment, no nucleotide is modified within 5nucleotides of the 5′ end of the 5′ extension domain, within 5nucleotides of the 3′ end of the 5′ extension domain, or within a regionthat is more than 5 nucleotides away from one or both ends of the 5′extension domain.

Modifications in the 5′ extension domain can be selected to notinterfere with gRNA molecule efficacy, which can be evaluated by testinga candidate modification in the system described in Section V. gRNA'shaving a candidate 5′ extension domain having a selected length,sequence, degree of complementarity, or degree of modification, can beevaluated in the system described at Section V. The candidate 5′extension domain can be placed, either alone, or with one or more othercandidate changes in a gRNA molecule/Cas9 molecule system known to befunctional with a selected target and evaluated.

In an embodiment, the 5′ extension domain has at least 60, 70, 80, 85,90 or 95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6nucleotides from, a reference 5′ extension domain, e.g., a naturallyoccurring, e.g., an S. pyogenes, S. aureus, or S. thermophilus, 5′extension domain, or a 5′ extension domain described herein, e.g., fromFIGS. 1A-1G.

Linking Domain

In a unimolecular gRNA molecule the linking domain is disposed betweenthe first and second complementarity domains. In a modular gRNAmolecule, the two molecules are associated with one another by thecomplementarity domains.

In an embodiment, the linking domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5,50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, inlength.

In an embodiment, the linking domain is 20+/−10, 30+/−10, 40+/−10,50+/−10, 60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, inlength.

In an embodiment, the linking domain is 10 to 100, 10 to 90, 10 to 80,10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15nucleotides in length. In other embodiments, the linking domain is 20 to100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to30, or 20 to 25 nucleotides in length.

In an embodiment, the linking domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16 17, 18, 19, or 20 nucleotides in length.

In an embodiment, the linking domain is a covalent bond.

In an embodiment, the linking domain comprises a duplexed region,typically adjacent to or within 1, 2, or 3 nucleotides of the 3′ end ofthe first complementarity domain and/or the 5-end of the secondcomplementarity domain. In an embodiment, the duplexed region can be20+/−10 base pairs in length. In an embodiment, the duplexed region canbe 10+/−5, 15+/−5, 20+/−5, or 30+/−5 base pairs in length. In anembodiment, the duplexed region can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 base pairs in length.

Typically the sequences forming the duplexed region have exactcomplementarity with one another, though in some embodiments as many as1, 2, 3, 4, 5, 6, 7 or 8 nucleotides are not complementary with thecorresponding nucleotides.

In an embodiment, the linking domain nucleotides do not comprisemodifications, e.g., modifications of the type provided in Section VIII.However, in an embodiment, the linking domain comprises one or moremodifications, e.g., modifications that it render it less susceptible todegradation or more bio-compatible, e.g., less immunogenic. By way ofexample, the backbone of the linking domain can be modified with aphosphorothioate, or other modification(s) from Section VIII. In anembodiment a nucleotide of the linking domain can comprise a 2′modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or othermodification(s) from Section VIII. In some embodiments, the linkingdomain can comprise as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications.

Modifications in a linking domain can be selected to not interfere withtargeting efficacy, which can be evaluated by testing a candidatemodification in the system described in Section V. gRNA's having acandidate linking domain having a selected length, sequence, degree ofcomplementarity, or degree of modification, can be evaluated a systemdescribed in Section V. A candidate linking domain can be placed, eitheralone, or with one or more other candidate changes in a gRNAmolecule/Cas9 molecule system known to be functional with a selectedtarget and evaluated.

In an embodiment, the linking domain has at least 60, 70, 80, 85, 90 or95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6nucleotides from, a reference linking domain, e.g., a linking domaindescribed herein, e.g., from FIGS. 1A-1G.

Proximal Domain

In an embodiment, the proximal domain is 6+/−2, 7+/−2, 8+/−2, 9+/−2,10+/−2, 11+/−2, 12+/−2, 13+/−2, 14+/−2, 14+/−2, 16+/−2, 17+/−2, 18+/−2,19+/−2, or 20+/−2 nucleotides in length.

In an embodiment, the proximal domain is 6, 7, 8, 9, 10, 11, 12, 13, 14,14, 16, 17, 18, 19, or 20 nucleotides in length.

In an embodiment, the proximal domain is 5 to 20, 7, to 18, 9 to 16, or10 to 14 nucleotides in length.

In an embodiment, the proximal domain nucleotides do not comprisemodifications, e.g., modifications of the type provided in Section VIII.However, in an embodiment, the proximal domain comprises one or moremodifications, e.g., modifications that it render it less susceptible todegradation or more bio-compatible, e.g., less immunogenic. By way ofexample, the backbone of the proximal domain can be modified with aphosphorothioate, or other modification(s) from Section VIII. In anembodiment a nucleotide of the proximal domain can comprise a 2′modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or othermodification(s) from Section VIII.

In some embodiments, the proximal domain can comprise as many as 1, 2,3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the proximal domaincomprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides ofits 5′ end, e.g., in a modular gRNA molecule. In an embodiment, thetarget domain comprises as many as 1, 2, 3, or 4 modifications within 5nucleotides of its 3′ end, e.g., in a modular gRNA molecule.

In some embodiments, the proximal domain comprises modifications at twoconsecutive nucleotides, e.g., two consecutive nucleotides that arewithin 5 nucleotides of the 5′ end of the proximal domain, within 5nucleotides of the 3′ end of the proximal domain, or more than 5nucleotides away from one or both ends of the proximal domain. In anembodiment, no two consecutive nucleotides are modified within 5nucleotides of the 5′ end of the proximal domain, within 5 nucleotidesof the 3′ end of the proximal domain, or within a region that is morethan 5 nucleotides away from one or both ends of the proximal domain. Inan embodiment, no nucleotide is modified within 5 nucleotides of the 5′end of the proximal domain, within 5 nucleotides of the 3′ end of theproximal domain, or within a region that is more than 5 nucleotides awayfrom one or both ends of the proximal domain.

Modifications in the proximal domain can be selected to not interferewith gRNA molecule efficacy, which can be evaluated by testing acandidate modification in the system described in Section V. gRNA'shaving a candidate proximal domain having a selected length, sequence,degree of complementarity, or degree of modification, can be evaluatedin the system described at Section V. The candidate proximal domain canbe placed, either alone, or with one or more other candidate changes ina gRNA molecule/Cas9 molecule system known to be functional with aselected target and evaluated.

In an embodiment, the proximal domain has at least 60, 70, 80, 85 90 or95% homology with, or differs by no more than 1, 2, 3, 4, 5, or 6nucleotides from, a reference proximal domain, e.g., a naturallyoccurring, e.g., an S. pyogenes, S. aureus, or S. thermophilus, proximaldomain, or a proximal domain described herein, e.g., from FIGS. 1A-1G.

Tail Domain

In an embodiment, the tail domain is 10+/−5, 20+/−5, 30+/−5, 40+/−5,50+/−5, 60+/−5, 70+/−5, 80+/−5, 90+/−5, or 100+/−5 nucleotides, inlength.

In an embodiment, the tail domain is 20+/−5 nucleotides in length.

In an embodiment, the tail domain is 20+/−10, 30+/−10, 40+/−10, 50+/−10,60+/−10, 70+/−10, 80+/−10, 90+/−10, or 100+/−10 nucleotides, in length.

In an embodiment, the tail domain is 25+/−10 nucleotides in length.

In an embodiment, the tail domain is 10 to 100, 10 to 90, 10 to 80, 10to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15nucleotides in length. In other embodiments, the tail domain is 20 to100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to30, or 20 to 25 nucleotides in length.

In an embodiment, the tail domain is 1 to 20, 1 to 1, 1 to 10, or 1 to 5nucleotides in length.

In an embodiment, the tail domain nucleotides do not comprisemodifications, e.g., modifications of the type provided in Section VIII.However, in an embodiment, the tail domain comprises one or moremodifications, e.g., modifications that it render it less susceptible todegradation or more bio-compatible, e.g., less immunogenic. By way ofexample, the backbone of the tail domain can be modified with aphosphorothioate, or other modification(s) from Section VIII. In anembodiment a nucleotide of the tail domain can comprise a 2′modification, e.g., a 2-acetylation, e.g., a 2′ methylation, or othermodification(s) from Section VIII.

In some embodiments, the tail domain can have as many as 1, 2, 3, 4, 5,6, 7 or 8 modifications. In an embodiment, the target domain comprisesas many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5′end. In an embodiment, the target domain comprises as many as 1, 2, 3,or 4 modifications within 5 nucleotides of its 3′ end.

In an embodiment, the tail domain comprises a tail duplex domain, whichcan form a tail duplexed region. In an embodiment, the tail duplexedregion can be 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 base pairs in length.In an embodiment, a further single stranded domain, exists 3′ to thetail duplexed domain. In an embodiment, this domain is 3, 4, 5, 6, 7, 8,9, or 10 nucleotides in length. In an embodiment it is 4 to 6nucleotides in length.

In an embodiment, the tail domain has at least 60, 70, 80, or 90%homology with, or differs by no more than 1, 2, 3, 4, 5, or 6nucleotides from, a reference tail domain, e.g., a naturally occurring,e.g., an S. pyogenes, or S. thermophilus, tail domain, or a tail domaindescribed herein, e.g., from FIGS. 1A-1G.

In an embodiment, the proximal and tail domain, taken together comprisethe following sequences:

(SEQ ID NO: 33) AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU, (SEQ IDNO: 34) AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGGUGC, (SEQ ID NO:35) AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCGGA UC, (SEQ ID NO:36) AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG, (SEQ ID NO: 37) AAGGCUAGUCCGUUAUCA,or (SEQ ID NO: 38) AAGGCUAGUCCG.

In an embodiment, the tail domain comprises the 3′ sequence UUUUUU,e.g., if a U6 promoter is used for transcription.

In an embodiment, the tail domain comprises the 3′ sequence UUUU, e.g.,if an H1 promoter is used for transcription.

In an embodiment, tail domain comprises variable numbers of 3′ Usdepending, e.g., on the termination signal of the pol-III promoter used.

In an embodiment, the tail domain comprises variable 3′ sequence derivedfrom the DNA template if a T7 promoter is used.

In an embodiment, the tail domain comprises variable 3′ sequence derivedfrom the DNA template, e.g., if in vitro transcription is used togenerate the RNA molecule.

In an embodiment, the tail domain comprises variable 3′ sequence derivedfrom the DNA template, e.g., if a pol-II promoter is used to drivetranscription.

Modifications in the tail domain can be selected to not interfere withtargeting efficacy, which can be evaluated by testing a candidatemodification in the system described in Section V. gRNAs having acandidate tail domain having a selected length, sequence, degree ofcomplementarity, or degree of modification, can be evaluated in thesystem described in Section V. The candidate tail domain can be placed,either alone, or with one or more other candidate changes in a gRNAmolecule/Cas9 molecule system known to be functional with a selectedtarget and evaluated.

In some embodiments, the tail domain comprises modifications at twoconsecutive nucleotides, e.g., two consecutive nucleotides that arewithin 5 nucleotides of the 5′ end of the tail domain, within 5nucleotides of the 3′ end of the tail domain, or more than 5 nucleotidesaway from one or both ends of the tail domain. In an embodiment, no twoconsecutive nucleotides are modified within 5 nucleotides of the 5′ endof the tail domain, within 5 nucleotides of the 3′ end of the taildomain, or within a region that is more than 5 nucleotides away from oneor both ends of the tail domain. In an embodiment, no nucleotide ismodified within 5 nucleotides of the 5′ end of the tail domain, within 5nucleotides of the 3′ end of the tail domain, or within a region that ismore than 5 nucleotides away from one or both ends of the tail domain.

In an embodiment a gRNA has the following structure:

5′ [targeting domain]-[first complementarity domain]-[linkingdomain]-[second complementarity domain]-[proximal domain]-[taildomain]-3′

wherein, the targeting domain comprises a core domain and optionally asecondary domain, and is 10 to 50 nucleotides in length;

the first complementarity domain is 5 to 25 nucleotides in length and,In an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homologywith a reference first complementarity domain disclosed herein;

the linking domain is 1 to 5 nucleotides in length;

the proximal domain is 5 to 20 nucleotides in length and, in anembodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with areference proximal domain disclosed herein; and

the tail domain is absent or a nucleotide sequence is 1 to 50nucleotides in length and, in an embodiment has at least 50, 60, 70, 80,85, 90 or 95% homology with a reference tail domain disclosed herein.

Exemplary Chimeric gRNAs

In an embodiment, a unimolecular, or chimeric, gRNA comprises,preferably from 5′ to 3′:

a targeting domain, e.g., comprising 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, or 26 nucleotides (which is complementary to a target nucleicacid);

a first complementarity domain;

a linking domain;

a second complementarity domain (which is complementary to the firstcomplementarity domain);

a proximal domain; and

a tail domain,

wherein,

(a) the proximal and tail domain, when taken together, comprise at least15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;

(b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53nucleotides 3′ to the last nucleotide of the second complementaritydomain; or

(c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54nucleotides 3′ to the last nucleotide of the second complementaritydomain that is complementary to its corresponding nucleotide of thefirst complementarity domain.

In an embodiment, the sequence from (a), (b), or (c), has at least 60,75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of anaturally occurring gRNA, or with a gRNA described herein.

In an embodiment, the proximal and tail domain, when taken together,comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53nucleotides.

In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45,49, 50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46,50, 51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of,16 nucleotides (e.g., 16 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 16 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,17 nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,18 nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,19 nucleotides (e.g., 19 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 19 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,20 nucleotides (e.g., 20 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 20 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,21 nucleotides (e.g., 21 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 21 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,22 nucleotides (e.g., 22 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 22 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,23 nucleotides (e.g., 23 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 23 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,24 nucleotides (e.g., 24 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 24 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,25 nucleotides (e.g., 25 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 25 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,26 nucleotides (e.g., 26 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 26 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,16 nucleotides (e.g., 16 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 16 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,16 nucleotides (e.g., 16 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 16 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,16 nucleotides (e.g., 16 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 16 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,17 nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,17 nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,17 nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,18 nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,18 nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,18 nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,19 nucleotides (e.g., 19 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 19 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,19 nucleotides (e.g., 19 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 19 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,19 nucleotides (e.g., 19 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 19 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,20 nucleotides (e.g., 20 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 20 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,20 nucleotides (e.g., 20 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 20 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,20 nucleotides (e.g., 20 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 20 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,21 nucleotides (e.g., 21 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 21 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,21 nucleotides (e.g., 21 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 21 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,21 nucleotides (e.g., 21 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 21 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,22 nucleotides (e.g., 22 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 22 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,22 nucleotides (e.g., 22 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 22 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,22 nucleotides (e.g., 22 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 22 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,23 nucleotides (e.g., 23 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 23 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,23 nucleotides (e.g., 23 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 23 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,23 nucleotides (e.g., 23 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 23 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,24 nucleotides (e.g., 24 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 24 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,24 nucleotides (e.g., 24 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 24 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,24 nucleotides (e.g., 24 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 24 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,25 nucleotides (e.g., 25 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 25 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,25 nucleotides (e.g., 25 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 25 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,25 nucleotides (e.g., 25 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 25 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,26 nucleotides (e.g., 26 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 26 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,26 nucleotides (e.g., 26 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 26 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,26 nucleotides (e.g., 26 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 26 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the unimolecular, or chimeric, gRNA molecule(comprising a targeting domain, a first complementary domain, a linkingdomain, a second complementary domain, a proximal domain and,optionally, a tail domain) comprises the following sequence in which thetargeting domain is depicted as 20 Ns but could be any sequence andrange in length from 16 to 26 nucleotides and in which the gRNA sequenceis followed by 6 Us, which serve as a termination signal for the U6promoter, but which could be either absent or fewer in number:

(SEQ ID NO: 45) NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUU UUUU.In an embodiment, the unimolecular, or chimeric, gRNA molecule is a S.pyogenes gRNA molecule.

In some embodiments, the unimolecular, or chimeric, gRNA molecule(comprising a targeting domain, a first complementary domain, a linkingdomain, a second complementary domain, a proximal domain and,optionally, a tail domain) comprises the following sequence in which thetargeting domain is depicted as 20 Ns but could be any sequence andrange in length from 16 to 26 nucleotides and in which the gRNA sequenceis followed by 6 Us, which serve as a termination signal for the U6promoter, but which could be either absent or fewer in number:

(SEQ ID NO: 2779) NNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUU UUUU(corresponding DNA sequence in SEQ ID NO: 2785). In an embodiment, theunimolecular, or chimeric, gRNA molecule is a S. aureus gRNA molecule.

The sequences and structures of exemplary chimeric gRNAs of SEQ ID NOs:45 and 2779 are shown in FIGS. 18A-18B, respectively.

Exemplary Modular gRNAs

In an embodiment, a modular gRNA comprises:

a first strand comprising, preferably from 5′ to 3′:

a targeting domain, e.g., comprising 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, or 26 nucleotides;

a first complementarity domain; and

a second strand, comprising, preferably from 5′ to 3′:

optionally a 5′ extension domain;

a second complementarity domain;

a proximal domain; and

a tail domain,

wherein:

(a) the proximal and tail domain, when taken together, comprise at least15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides;

(b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53nucleotides 3′ to the last nucleotide of the second complementaritydomain; or

(c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54nucleotides 3′ to the last nucleotide of the second complementaritydomain that is complementary to its corresponding nucleotide of thefirst complementarity domain.

In an embodiment, the sequence from (a), (b), or (c), has at least 60,75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of anaturally occurring gRNA, or with a gRNA described herein.

In an embodiment, the proximal and tail domain, when taken together,comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53nucleotides.

In an embodiment, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45,49, 50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46,50, 51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (e.g., 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) havingcomplementarity with the target domain, e.g., the targeting domain is16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

In an embodiment, the targeting domain comprises, has, or consists of,16 nucleotides (e.g., 16 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 16 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,17 nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,18 nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,19 nucleotides (e.g., 19 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 19 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,20 nucleotides (e.g., 20 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 20 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,21 nucleotides (e.g., 21 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 21 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,22 nucleotides (e.g., 22 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 22 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,23 nucleotides (e.g., 23 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 23 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,24 nucleotides (e.g., 24 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 24 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,25 nucleotides (e.g., 25 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 25 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,26 nucleotides (e.g., 26 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 26 nucleotides inlength.

In an embodiment, the targeting domain comprises, has, or consists of,16 nucleotides (e.g., 16 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 16 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,16 nucleotides (e.g., 16 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 16 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,16 nucleotides (e.g., 16 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 16 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,17 nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,17 nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,17 nucleotides (e.g., 17 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 17 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,18 nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,18 nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,18 nucleotides (e.g., 18 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 18 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,19 nucleotides (e.g., 19 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 19 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,19 nucleotides (e.g., 19 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 19 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,19 nucleotides (e.g., 19 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 19 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,20 nucleotides (e.g., 20 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 20 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,20 nucleotides (e.g., 20 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 20 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,20 nucleotides (e.g., 20 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 20 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,21 nucleotides (e.g., 21 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 21 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,21 nucleotides (e.g., 21 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 21 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,21 nucleotides (e.g., 21 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 21 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,22 nucleotides (e.g., 22 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 22 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,22 nucleotides (e.g., 22 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 22 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,22 nucleotides (e.g., 22 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 22 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,23 nucleotides (e.g., 23 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 23 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,23 nucleotides (e.g., 23 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 23 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,23 nucleotides (e.g., 23 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 23 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,24 nucleotides (e.g., 24 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 24 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,24 nucleotides (e.g., 24 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 24 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,24 nucleotides (e.g., 24 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 24 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,25 nucleotides (e.g., 25 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 25 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,25 nucleotides (e.g., 25 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 25 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,25 nucleotides (e.g., 25 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 25 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,26 nucleotides (e.g., 26 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 26 nucleotides inlength; and the proximal and tail domain, when taken together, compriseat least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides.

In an embodiment, the targeting domain comprises, has, or consists of,26 nucleotides (e.g., 26 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 26 nucleotides inlength; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,50, or 53 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain.

In an embodiment, the targeting domain comprises, has, or consists of,26 nucleotides (e.g., 26 consecutive nucleotides) having complementaritywith the target domain, e.g., the targeting domain is 26 nucleotides inlength; and there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,51, or 54 nucleotides 3′ to the last nucleotide of the secondcomplementarity domain that is complementary to its correspondingnucleotide of the first complementarity domain.

gRNA Modifications

The activity, stability, or other characteristics of gRNAs can bealtered through the incorporation of chemical and/or sequentialmodifications. As one example, transiently expressed or deliverednucleic acids can be prone to degradation by, e.g., cellular nucleases.Accordingly, the gRNAs described herein can contain one or more modifiednucleosides or nucleotides which introduce stability toward nucleases.While not wishing to be bound by theory it is also believed that certainmodified gRNAs described herein can exhibit a reduced innate immuneresponse when introduced into a population of cells, particularly thecells of the present invention. As noted above, the term “innate immuneresponse” includes a cellular response to exogenous nucleic acids,including single stranded nucleic acids, generally of viral or bacterialorigin, which involves the induction of cytokine expression and release,particularly the interferons, and cell death.

One common 3′ end modification is the addition of a poly A tractcomprising one or more (and typically 5-200) adenine (A) residues. Thepoly A tract can be contained in the nucleic acid sequence encoding thegRNA, or can be added to the gRNA during chemical synthesis, orfollowing in vitro transcription using a polyadenosine polymerase (e.g.,E. coli Poly(A)Polymerase). In vivo, poly-A tracts can be added tosequences transcribed from DNA vectors through the use ofpolyadenylation signals. Examples of such signals are provided inMaeder.

III. Methods for Designing gRNAs

Methods for designing gRNAs are described herein, including methods forselecting, designing and validating target domains. Exemplary targetingdomains are also provided herein. Targeting Domains discussed herein canbe incorporated into the gRNAs described herein.

Methods for selection and validation of target sequences as well asoff-target analyses are described, e.g., in Mali 2013; Hsu 2013; Fu2014; Heigwer 2014; Bae 2014; Xiao 2014.

For example, a software tool can be used to optimize the choice of gRNAwithin a user's target sequence, e.g., to minimize total off-targetactivity across the genome. Off target activity may be other thancleavage. For each possible gRNA choice using S. pyogenes Cas9, softwaretools can identify all potential off-target sequences (preceding eitherNAG or NGG PAMs) across the genome that contain up to a certain number(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. Thecleavage efficiency at each off-target sequence can be predicted, e.g.,using an experimentally-derived weighting scheme. Each possible gRNA canthen ranked according to its total predicted off-target cleavage; thetop-ranked gRNAs represent those that are likely to have the greateston-target and the least off-target cleavage. Other functions, e.g.,automated reagent design for gRNA vector construction, primer design forthe on-target Surveyor assay, and primer design for high-throughputdetection and quantification of off-target cleavage via next-generationsequencing, can also be included in the tool. Candidate gRNA moleculescan be evaluated by art-known methods or as described in Section Vherein.

Guide RNAs (gRNAs) for use with S. pyogenes, S. aureus and N.meningitidis Cas9s were identified using a DNA sequence searchingalgorithm. Guide RNA design was carried out using a custom guide RNAdesign software based on the public tool cas-offinder (Bae 2014). Saidcustom guide RNA design software scores guides after calculating theirgenomewide off-target propensity. Typically matches ranging from perfectmatches to 7 mismatches are considered for guides ranging in length from17 to 24. Once the off-target sites are computationally determined, anaggregate score is calculated for each guide and summarized in a tabularoutput using a web-interface. In addition to identifying potential gRNAsites adjacent to PAM sequences, the software also identifies all PAMadjacent sequences that differ by 1, 2, 3 or more nucleotides from theselected gRNA sites. Genomic DNA sequence for each gene was obtainedfrom the UCSC Genome browser and sequences were screened for repeatelements using the publically available RepeatMasker program.RepeatMasker searches input DNA sequences for repeated elements andregions of low complexity. The output is a detailed annotation of therepeats present in a given query sequence.

Following identification, gRNAs were ranked into tiers based on theirdistance to the target site, their orthogonality and presence of a 5′ G(based on identification of close matches in the human genome containinga relevant PAM, e.g., in the case of S. pyogenes, a NGG PAM, in the caseof S. aureus, NNGRR (e.g., a NNGRRT or NNGRRV) PAM, and in the case ofN. meningitides, a NNNNGATT or NNNNGCTT PAM. Orthogonality refers to thenumber of sequences in the human genome that contain a minimum number ofmismatches to the target sequence. A “high level of orthogonality” or“good orthogonality” may, for example, refer to 20-mer gRNAs that haveno identical sequences in the human genome besides the intended target,nor any sequences that contain one or two mismatches in the targetsequence. Targeting domains with good orthogonality are selected tominimize off-target DNA cleavage.

As an example, for S. pyogenes and N. meningitides targets, 17-mer, or20-mer gRNAs were designed. As another example, for S. aureus targets,18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer and 24-mer gRNAs weredesigned. Targeting domains, disclosed herein, may comprises the 17-merdescribed in Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D,Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B,or Table 11, e.g., the targeting domains of 18 or more nucleotides maycomprise the 17-mer gRNAs described in Tables 2A-2D, Tables 3A-3C,Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D,Tables 9A-9E, Tables 10A-10B, or Table 11. Targeting domains, disclosedherein, may comprises the 18-mer described in Tables 2A-2D, Tables3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11, e.g., the targetingdomains of 19 or more nucleotides may comprise the 18-mer gRNAsdescribed in Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D,Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B,or Table 11. Targeting domains, disclosed herein, may comprises the19-mer described in Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables10A-10B, or Table 11, e.g., the targeting domains of 20 or morenucleotides may comprise the 19-mer gRNAs described in Tables 2A-2D,Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D,Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11. Targetingdomains, disclosed herein, may comprises the 20-mer gRNAs described inTables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B,Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11,e.g., the targeting domains of 21 or more nucleotides may comprise the20-mer gRNAs described in Tables 2A-2D, Tables 3A-3C, Tables 4A-4D,Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E,Tables 10A-10B, or Table 11. Targeting domains, disclosed herein, maycomprises the 21-mer described in Tables 2A-2D, Tables 3A-3C, Tables4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables9A-9E, Tables 10A-10B, or Table 11, e.g., the targeting domains of 22 ormore nucleotides may comprise the 21-mer gRNAs described in Tables2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11.Targeting domains, disclosed herein, may comprises the 22-mer describedin Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B,Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11,e.g., the targeting domains of 23 or more nucleotides may comprise the22-mer gRNAs described in Tables 2A-2D, Tables 3A-3C, Tables 4A-4D,Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E,Tables 10A-10B, or Table 11. Targeting domains, disclosed herein, maycomprises the 23-mer described in Tables 2A-2D, Tables 3A-3C, Tables4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables9A-9E, Tables 10A-10B, or Table 11, e.g., the targeting domains of 24 ormore nucleotides may comprise the 23-mer gRNAs described in Tables2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11.Targeting domains, disclosed herein, may comprises the 24-mer describedin Tables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B,Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11,e.g., the targeting domains of 25 or more nucleotides may comprise the24-mer gRNAs described in Tables 2A-2D, Tables 3A-3C, Tables 4A-4D,Tables 5A-5D, Tables 6A-6B, Tables 7A-7D, Tables 8A-8D, Tables 9A-9E,Tables 10A-10B, or Table 11.

gRNAs were identified for both single-gRNA nuclease cleavage and for adual-gRNA paired “nickase” strategy. Criteria for selecting gRNAs andthe determination for which gRNAs can be used for the dual-gRNA paired“nickase” strategy is based on two considerations:

-   -   1. gRNA pairs should be oriented on the DNA such that PAMs are        facing out and cutting with the D10A Cas9 nickase will result in        5′ overhangs.    -   2. An assumption that cleaving with dual nickase pairs will        result in deletion of the entire intervening sequence at a        reasonable frequency. However, cleaving with dual nickase pairs        can also result in indel mutations at the site of only one of        the gRNAs. Candidate pair members can be tested for how        efficiently they remove the entire sequence versus causing indel        mutations at the site of one gRNA.

The Targeting Domains discussed herein can be incorporated into thegRNAs described herein.

Three strategies were utilized to identify gRNAs for use with S.pyogenes, S. aureus and N. meningitidis Cas9 enzymes.

In one strategy, gRNAs were designed for use with S. pyogenes and S.aureus Cas9 enzymes to induce an indel mediated by NHEJ in closeproximity to or including the LCA10 target position (e.g., c.2991+1655Ato G). The gRNAs were identified and ranked into 4 tiers for S. pyogenes(Tables 2A-2D). The targeting domain for tier 1 gRNA molecules to beused with S. pyogenes Cas9 molecules were selected based on (1) a shortdistance to the target position, e.g., within 40 bp upstream and 40 bpdownstream of the mutation, (2) a high level of orthogonality, and (3)the presence of a 5′ G. For selection of tier 2 gRNAs, a short distanceand high orthogonality were required but the presence of a 5′G was notrequired. Tier 3 uses the same distance restriction and the requirementfor a 5′G, but removes the requirement of good orthogonality. Tier 4uses the same distance restriction but removes the requirement of goodorthogonality and the 5′G. The gRNAs were identified and ranked into 4tiers for S. aureus, when the relevant PAM was NNGRR (Tables 3A-3C). Thetargeting domain for tier 1 gRNA molecules to be used with S. pyogenesCas9 molecules were selected based on (1) a short distance to the targetposition, e.g., within 40 bp upstream and 40 bp downstream of themutation, (2) a high level of orthogonality, and (3) the presence of a5′ G. For selection of tier 2 gRNAs, a short distance and highorthogonality were required but the presence of a 5′G was not required.Tier 3 uses the same distance restriction and the requirement for a 5′G,but removes the requirement of good orthogonality. Tier 4 uses the samedistance restriction but removes the requirement of good orthogonalityand the 5′G. The gRNAs were identified and ranked into 5 tiers for S.aureus when the relevant PAM was NNGRRT or NNGRRV (Tables 7A-7D). Thetargeting domain for tier 1 gRNA molecules to be used with S. aureusCas9 molecules were selected based on (1) a short distance to the targetposition, e.g., within 40 bp upstream and 40 bp downstream of themutation, (2) a high level of orthogonality, (3) the presence of a 5′ Gand (4) PAM was NNGRRT. For selection of tier 2 gRNAs, a short distanceand high orthogonality were required but the presence of a 5′G was notrequired, and PAM was NNGRRT. Tier 3 uses the same distance restrictionand the requirement for a 5′G, but removes the requirement of goodorthogonality, and PAM was NNGRRT. Tier 4 uses the same distancerestriction but removes the requirement of good orthogonality and the5′G, and PAM was NNGRRT. Tier 5 required a short distance to the targetposition, e.g., within 40 bp upstream and 40 bp downstream of themutation and PAM was NNGRRV. Note that tiers are non-inclusive (eachgRNA is listed only once for the strategy). In certain instances, nogRNA was identified based on the criteria of the particular tier.

In a second strategy, gRNAs were designed for use with S. pyogenes, S.aureus and N. meningitidis Cas9 molecules to delete a genomic sequenceincluding the mutation at the LCA10 target position (e.g., c.2991+1655Ato G), e.g., mediated by NHEJ. The gRNAs were identified and ranked into4 tiers for S. pyogenes (Tables 4A-4D). The targeting domain to be usedwith S. pyogenes Cas9 molecules for tier 1 gRNA molecules were selectedbased on (1) flanking the mutation without targeting unwanted chromosomeelements, such as an Alu repeat, e.g., within 400 bp upstream of an Alurepeat or 700 bp downstream of mutation, (2) a high level oforthogonality, and (3) the presence of a 5′ G. For selection of tier 2gRNAs, a reasonable distance and high orthogonality were required butthe presence of a 5′G was not required. Tier 3 uses the same distancerestriction and the requirement for a 5′G, but removes the requirementof good orthogonality. Tier 4 uses the same distance restriction butremoves the requirement of good orthogonality and the 5′G. The gRNAswere identified and ranked into 4 tiers for S. aureus, when the relevantPAM was NNGRR (Tables 5A-5D). The targeting domain to be used with S.aureus Cas9 molecules for tier 1 gRNA molecules were selected based on(1) flanking the mutation without targeting unwanted chromosomeelements, such as an Alu repeat, e.g., within 400 bp upstream of an Alurepeat or 700 bp downstream of mutation, (2) a high level oforthogonality, and (3) the presence of a 5′ G. For selection of tier 2gRNAs, a reasonable distance and high orthogonality were required butthe presence of a 5′G was not required. Tier 3 uses the same distancerestriction and the requirement for a 5′G, but removes the requirementof good orthogonality. Tier 4 uses the same distance restriction butremoves the requirement of good orthogonality and the 5′G. The gRNAswere identified and ranked into 2 tiers for N. meningitides (Tables6A-6B). The targeting domain to be used with N. meningitides Cas9molecules for tier 1 gRNA molecules were selected based on (1) flankingthe mutation without targeting unwanted chromosome elements, such as anAlu repeat, e.g., within 400 bp upstream of an Alu repeat or 700 bpdownstream of mutation, (2) a high level of orthogonality, and (3) thepresence of a 5′ G. For selection of tier 2 gRNAs, a reasonable distanceand high orthogonality were required but the presence of a 5′G was notrequired. Note that tiers are non-inclusive (each gRNA is listed onlyonce for the strategy). In certain instances, no gRNA was identifiedbased on the criteria of the particular tier. In a third strategy, gRNAswere designed for use with S. pyogenes, S. aureus and N. meningitidisCas9 molecules to delete a genomic sequence including the mutation atthe LCA10 target position (e.g., c.2991+1655A to G), e.g., mediated byNHEJ. The gRNAs were identified and ranked into 4 tiers for S. pyogenes(Tables 8A-8D). The targeting domain to be used with S. pyogenes Cas9enzymes for tier 1 gRNA molecules were selected based on (1) flankingthe mutation without targeting unwanted chromosome elements, such as anAlu repeat, e.g., within 1000 bp upstream of an Alu repeat or 1000 bpdownstream of mutation, (2) a high level of orthogonality, (3) thepresence of a 5′ G and (4) and PAM was NNGRRT. For selection of tier 2gRNAs, a reasonable distance and high orthogonality were required butthe presence of a 5′G was not required, and PAM was NNGRRT. Tier 3 usesthe same distance restriction and the requirement for a 5′G, but removesthe requirement of good orthogonality, and PAM was NNGRRT. Tier 4 usesthe same distance restriction but removes the requirement of goodorthogonality and the 5′G, and PAM was NNGRRT. The gRNAs were identifiedand ranked into 4 tiers for S. aureus, when the relevant PAM was NNGRRTor NNGRRV (Tables 9A-9E). The targeting domain to be used with S. aureusCas9 enzymes for tier 1 gRNA molecules were selected based on (1)flanking the mutation without targeting unwanted chromosome elements,such as an Alu repeat, e.g., within 1000 bp upstream of an Alu repeat or1000 bp downstream of mutation, (2) a high level of orthogonality, and(3) the presence of a 5′ G. For selection of tier 2 gRNAs, a reasonabledistance and high orthogonality were required but the presence of a 5′Gwas not required. Tier 3 uses the same distance restriction and therequirement for a 5′G, but removes the requirement of goodorthogonality. Tier 4 uses the same distance restriction but removes therequirement of good orthogonality and the 5′G. Tier 5 used the samedistance restriction and PAM was NNGRRV. The gRNAs were identified andranked into 2 tiers for N. meningitides (Tables 10A-10B). The targetingdomain to be used with N. meningitides Cas9 molecules for tier 1 gRNAmolecules were selected based on (1) flanking the mutation withouttargeting unwanted chromosome elements, such as an Alu repeat, e.g.,within 1000 bp upstream of an Alu repeat or 1000 bp downstream ofmutation, (2) a high level of orthogonality, and (3) the presence of a5′ G. For selection of tier 2 gRNAs, a reasonable distance and highorthogonality were required but the presence of a 5′G was not required.Note that tiers are non-inclusive (each gRNA is listed only once for thestrategy). In certain instances, no gRNA was identified based on thecriteria of the particular tier.

In an embodiment, when a single gRNA molecule is used to target a Cas9nickase to create a single strand break to introduce a break-inducedindel in close proximity to or including the LCA10 target position, thegRNA is used to target either upstream of (e.g., within 40 bp upstreamof the LCA10 target position), or downstream of (e.g., within 40 bpdownstream of the LCA10 target position) in the CEP290 gene.

In an embodiment, when a single gRNA molecule is used to target a Cas9nuclease to create a double strand break to introduce a break-inducedindel in close proximity to or including the LCA10 target position, thegRNA is used to target either upstream of (e.g., within 40 bp upstreamof the LCA10 target position), or downstream of (e.g., within 40 bpdownstream of the LCA10 target position) in the CEP290 gene.

In an embodiment, dual targeting is used to create two double strandbreaks to delete a genomic sequence including the mutation at the LCA10target position, e.g., mediated by NHEJ. In an embodiment, the first andsecond gRNAs are used target two Cas9 nucleases to flank, e.g., thefirst of gRNA is used to target upstream of (e.g., within 400 bpupstream of the Alu repeat, or within 40 bp upstream of the LCA10 targetposition), and the second gRNA is used to target downstream of (e.g.,within 700 bp downstream of the LCA10 target position) in the CEP290gene.

In an embodiment, dual targeting is used to create a double strand breakand a pair of single strand breaks to delete a genomic sequenceincluding the mutation at the LCA10 target position, e.g., mediated byNHEJ. In an embodiment, the first, second and third gRNAs are used totarget one Cas9 nuclease and two Cas9 nickases to flank, e.g., the firstgRNA that will be used with the Cas9 nuclease is used to target upstreamof (e.g., within 400 bp upstream of the Alu repeat, or within 40 bpupstream of the LCA10 target position) or downstream of (e.g., within700 bp downstream) of the LCA10 target position, and the second andthird gRNAs that will be used with the Cas9 nickase pair are used totarget the opposite side of the LCA10 target position (e.g., within 400bp upstream of the Alu repeat, within 40 bp upstream of the LCA10 targetposition, or within 700 bp downstream of the LCA10 target position) inthe CEP290 gene.

In an embodiment, when four gRNAs (e.g., two pairs) are used to targetfour Cas9 nickases to create four single strand breaks to delete genomicsequence including the mutation at the LCA10 target position, e.g.,mediated by NHEJ, the first pair and second pair of gRNAs are used totarget four Cas9 nickases to flank, e.g., the first pair of gRNAs areused to target upstream of (e.g., within 400 bp upstream of the Alurepeat, or within 40 bp upstream of the LCA10 target position), and thesecond pair of gRNAs are used to target downstream of (e.g., within 700bp downstream of the LCA10 target position) in the CEP290 gene.

In an embodiment, dual targeting is utilized to delete genomic sequenceincluding the mutation at the LCA10 target position mediated by NHEJ. Itis contemplated herein that in an embodiment any upstream gRNA (e.g.,within 400 bp upstream of an Alu repeat, or within 40 bp upstream of theLCA10 target position) in Tables 2A-2C and Tables 4A-4D can be pairedwith any downstream gRNA (e.g., within 700 downstream of LCA10 targetposition) in Tables 4A-4D to be used with a S. pyogenes Cas9 molecule togenerate dual targeting. Exemplary pairs including selecting a targetingdomain that is labeled as upstream from Tables 2A-2C or Tables 4A-4D anda second targeting domain that is labeled as downstream from Tables4A-4D. In an embodiment, a targeting domain that is labeled as upstreamin Tables 2A-2C or Tables 4A-4D can be combined with any of thetargeting domains that is labeled as downstream in Tables 4A-4D.

In an embodiment, dual targeting is utilized to delete genomic sequenceincluding the mutation at the LCA10 target position mediated by NHEJ. Itis contemplated herein that in an embodiment any upstream gRNA (e.g.,within 400 bp upstream of an Alu repeat, or within 40 bp upstream of theLCA10 target position) in Tables 3A-3C and Tables 5A-5D can be pairedwith any downstream gRNA (e.g., within 700 downstream of LCA10 targetposition) in Tables 5A-5D to be used with a S. aureus Cas9 molecule togenerate dual targeting. Exemplary pairs include selecting a targetingdomain that is labeled as upstream from Tables 3A-3C or Tables 5A-5D anda second targeting domain that is labeled as downstream from Tables5A-5D. In an embodiment, a targeting domain that is labeled as upstreamin Tables 3A-3C or Tables 5A-5D can be combined with any of thetargeting domains that is labeled as downstream in Tables 5A-5D.

In an embodiment, dual targeting is utilized to delete genomic sequenceincluding the mutation at the LCA10 target position mediated by NHEJ. Itis contemplated herein that in an embodiment any upstream gRNA (e.g.,within 400 bp upstream of an Alu repeat, or within 40 bp upstream of theLCA10 target position) in Tables 6A-6B can be paired with any downstreamgRNA (e.g., within 700 downstream of LCA10 target position) in Tables6A-6B to be used with a N. meningitidis Cas9 molecule to generate dualtargeting. Exemplary pairs include selecting a targeting domain that islabeled as upstream from Tables 6A-6B and a second targeting domain thatis labeled as downstream from Tables 6A-6B. In an embodiment, atargeting domain that is labeled as upstream in Tables 6A-6B can becombined with any of the targeting domains that is labeled as downstreamin Tables 6A-6B.

In an embodiment, dual targeting (e.g., dual double strand cleavage) isused to create two double strand breaks to delete a genomic sequenceincluding the mutation at the LCA10 target position, e.g., mediated byNHEJ. In an embodiment, the first and second gRNAs are used target twoCas9 nucleases to flank, e.g., the first of gRNA is used to targetupstream of (e.g., within 1000 bp upstream of the Alu repeat, or within40 bp upstream of the LCA10 target position), and the second gRNA isused to target downstream of (e.g., within 1000 bp downstream of theLCA10 target position) in the CEP290 gene.

In an embodiment, dual targeting (e.g., dual double strand cleavage) isused to create a double strand break and a pair of single strand breaksto delete a genomic sequence including the mutation at the LCA10 targetposition, e.g., mediated by NHEJ. In an embodiment, the first, secondand third gRNAs are used to target one Cas9 nuclease and two Cas9nickases to flank, e.g., the first gRNA that will be used with the Cas9nuclease is used to target upstream of (e.g., within 1000 bp upstream ofthe Alu repeat, or within 40 bp upstream of the LCA10 target position)or downstream of (e.g., within 1000 bp downstream) of the LCA10 targetposition, and the second and third gRNAs that will be used with the Cas9nickase pair are used to target the opposite side of the LCA10 targetposition (e.g., within 1000 bp upstream of the Alu repeat, or within 40bp upstream of the LCA10 target position or within 1000 bp downstream ofthe LCA10 target position) in the CEP290 gene.

In an embodiment, when four gRNAs (e.g., two pairs) are used to targetfour Cas9 nickases to create four single strand breaks to delete genomicsequence including the mutation at the LCA10 target position, e.g.,mediated by NHEJ, the first pair and second pair of gRNAs are used totarget four Cas9 nickases to flank, e.g., the first pair of gRNAs areused to target upstream of (e.g., within 1000 bp upstream of the Alurepeat, or within 40 bp upstream of the LCA10 target position), and thesecond pair of gRNAs are used to target downstream of (e.g., within 1000bp downstream of the LCA10 target position) in the CEP290 gene.

In an embodiment, dual targeting is utilized to delete genomic sequenceincluding the mutation at the LCA10 target position, e.g., mediated byNHEJ. It is contemplated herein that in an embodiment any upstream gRNA(e.g., within 1000 bp upstream of an Alu repeat, or within 40 bpupstream of the LCA10 target position) in Tables 2A-2C, Tables 4A-4D, orTables 8A-8D can be paired with any downstream gRNA (e.g., within 1000downstream of LCA10 target position) in Tables 2A-2C, Tables 4A-4D, orTables 8A-8D to be used with a S. pyogenes Cas9 molecule to generatedual targeting. Exemplary pairs including selecting a targeting domainthat is labeled as upstream from Tables 2A-2C, Tables 4A-4D, or Tables8A-8D and a second targeting domain that is labeled as downstream fromTables 2A-2C, Tables 4A-4D, or Tables 8A-8D. In an embodiment, atargeting domain that is labeled as upstream in Tables 2A-2C, Tables4A-4D, or Tables 8A-8D can be combined with any of the targeting domainsthat is labeled as downstream in Tables 2A-2C, Tables 4A-4D, or Tables8A-8D.

In an embodiment, dual targeting is utilized to delete genomic sequenceincluding the mutation at the LCA10 target position mediated by NHEJ. Itis contemplated herein that in an embodiment any upstream gRNA (e.g.,within 1000 bp upstream of an Alu repeat, or within 40 bp upstream ofthe LCA10 target position) in Tables 3A-3C, Tables 5A-5D, Tables 7A-7D,or Tables 9A-9E can be paired with any downstream gRNA (e.g., within1000 downstream of LCA10 target position) in Tables 3A-3C, Tables 5A-5D,Tables 7A-7D, or Tables 9A-9E to be used with a S. aureus Cas9 moleculeto generate dual targeting. Exemplary pairs include selecting atargeting domain that is labeled as upstream from Tables 3A-3C, Tables5A-5D, Tables 7A-7D, or Tables 9A-9E and a second targeting domain thatis labeled as downstream from Tables 3A-3C, Tables 5A-5D, Tables 7A-7D,or Tables 9A-9E. In an embodiment, a targeting domain that is labeled asupstream in Tables 3A-3C, Tables 5A-5D, Tables 7A-7D, or Tables 9A-9Ecan be combined with any of the targeting domains that is labeled asdownstream in Tables 3A-3C, Tables 5A-5D, Tables 7A-7D, or Tables 9A-9E.

In an embodiment, dual targeting is utilized to delete genomic sequenceincluding the mutation at the LCA10 target position, e.g., mediated byNHEJ. It is contemplated herein that in an embodiment any upstream gRNA(e.g., within 1000 bp upstream of an Alu repeat, or within 40 bpupstream of the LCA10 target position) in Tables 6A-6B or Tables 10A-10Bcan be paired with any downstream gRNA (e.g., within 1000 downstream ofLCA10 target position) in Tables 6A-6D to be used with a N. meningitidisCas9 molecule to generate dual targeting. Exemplary pairs includeselecting a targeting domain that is labeled as upstream from Tables6A-6B or Tables 10A-10B and a second targeting domain that is labeled asdownstream from Tables 6A-6B or Tables 10A-10B. In an embodiment, atargeting domain that is labeled as upstream in Tables 6A-6B or Tables10A-10B and can be combined with any of the targeting domains that islabeled as downstream in Tables 6A-6B or Tables 10A-10B.

Any of the targeting domains in the tables described herein can be usedwith a Cas9 nickase molecule to generate a single strand break.

Any of the targeting domains in the tables described herein can be usedwith a Cas9 nuclease molecule to generate a double strand break.

In an embodiment, dual targeting (e.g., dual nicking) is used to createtwo nicks on opposite DNA strands by using S. pyogenes, S. aureus and N.meningitidis Cas9 nickases with two targeting domains that arecomplementary to opposite DNA strands, e.g., a gRNA comprising any minusstrand targeting domain may be paired any gRNA comprising a plus strandtargeting domain provided that the two gRNAs are oriented on the DNAsuch that PAMs face outward and the distance between the 5′ ends of thegRNAs is 0-50 bp. Exemplary nickase pairs including selecting atargeting domain from Group A and a second targeting domain from Group Bin Table 2D (for S. pyogenes), or selecting a targeting domain fromGroup A and a second targeting domain from Group B in Table 7D (for S.aureus). It is contemplated herein that in an embodiment a targetingdomain of Group A can be combined with any of the targeting domains ofGroup B in Table 2D (for S. pyogenes). For example, CEP290-B5 orCEP290-B10 can be combined with CEP290-B1 or CEP290-B6. It iscontemplated herein that in an embodiment a targeting domain of Group Acan be combined with any of the targeting domains of Group B in Table 7D(for S. aureus). For example, CEP290-12 or CEP290-17 can be combinedwith CEP290-11 or CEP290-16.

In an embodiment, dual targeting (e.g., dual double strand cleavage) isused to create two double strand breaks by using S. pyogenes, S. aureusand N. meningitidis Cas9 nucleases with two targeting domains. It iscontemplated herein that in an embodiment any upstream gRNA of any ofTables 2A-2C, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B,Tables 7A-7C, Tables 8A-8D, Tables 9A-9E, or Tables 10A-10B can bepaired with any downstream gRNA of any of Tables 2A-2C, Tables 3A-3C,Tables 4A-4D, Tables 5A-5D, Tables 6A-6B, Tables 7A-7C, Tables 8A-8D,Tables 9A-9E, or Tables 10A-10B. Exemplary nucleases pairs are shown inTable 11, e.g., CEP290-323 can be combined with CEP290-11, CEP290-323can be combined with CEP290-64, CEP290-490 can be combined withCEP290-496, CEP290-490 can be combined with CEP290-502, CEP290-490 canbe combined with CEP290-504, CEP290-492 can be combined with CEP290-502,or CEP290-492 can be combined with CEP290-504.

It is contemplated herein that any upstream gRNA described herein may bepaired with any downstream gRNA described herein. When an upstream gRNAdesigned for use with one species of Cas9 is paired with a downstreamgRNA designed for use from a different species of Cas9, both Cas9species are used to generate a single or double-strand break, asdesired.

Exemplary Targeting Domains

Table 2A provides targeting domains for NHEJ-mediated introduction of anindel in close proximity to or including the LCA10 target position inthe CEP290 gene selected according to the first tier parameters. Thetargeting domains are within 40 bases of the LCA10 target position, havegood orthogonality, and start with G. It is contemplated herein that thetargeting domain hybridizes to the target domain through complementarybase pairing. Any of the targeting domains in the table can be used witha S. pyogenes Cas9 molecule that generates a double stranded break (Cas9nuclease) or a single-stranded break (Cas9 nickase).

TABLE 2A Target DNA Site Position relative gRNA Name Strand TargetingDomain Length to mutation CEP290-B4 + GAGAUACUCACAAUUACAAC 20 upstream(SEQ ID NO: 395) CEP290-B28 + GAUACUCACAAUUACAACUG 20 upstream (SEQ IDNO: 396)

Table 2B provides targeting domains for NHEJ-mediated introduction of anindel in close proximity to or including the LCA10 target position inthe CEP290 gene selected according to the second tier parameters. Thetargeting domains are within 40 bases of the LCA10 target position, havegood orthogonality, and do not start with G. It is contemplated hereinthat the targeting domain hybridizes to the target domain throughcomplementary base pairing. Any of the targeting domains in the tablecan be used with a S. pyogenes Cas9 molecule that generates a doublestranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 2B Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B6 − CUCAUACCUAUCCCUAU 17 downstream (SEQID NO: 594) CEP290-B20 + ACACUGCCAAUAGGGAU 17 downstream (SEQ ID NO:595) CEP290-B10 + CAAUUACAACUGGGGCC 17 upstream (SEQ ID NO: 596)CEP290-B21 + CUAAGACACUGCCAAUA 17 downstream (SEQ ID NO: 597)CEP290-B9 + AUACUCACAAUUACAAC 17 upstream (SEQ ID NO: 598) CEP290-B1 −UAUCUCAUACCUAUCCCUAU 20 downstream (SEQ ID NO: 599) CEP290-B29 +AAGACACUGCCAAUAGGGAU 20 downstream (SEQ ID NO: 600) CEP290-B5 +UCACAAUUACAACUGGGGCC 20 upstream (SEQ ID NO: 601) CEP290-B30 +AGAUACUCACAAUUACAACU 20 upstream (SEQ ID NO: 602)

Table 2C provides targeting domains for NHEJ-mediated introduction of anindel in close proximity to or including the LCA10 target position inthe CEP290 gene selected according to the fourth tier parameters. Thetargeting domains are within 40 bases of the LCA10 target position anddo not start with G. It is contemplated herein that the targeting domainhybridizes to the target domain through complementary base pairing. Anyof the targeting domains in the table can be used with a S. pyogenesCas9 molecule that generates a double stranded break (Cas9 nuclease) ora single-stranded break (Cas9 nickase).

TABLE 2C Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B22 + ACUAAGACACUGCCAAU (SEQ 17 downstreamID NO: 603) CEP290-B23 + UACUCACAAUUACAACU (SEQ 17 upstream ID NO: 604)CEP290-B24 + ACUCACAAUUACAACUG (SEQ 17 upstream ID NO: 605) CEP290-B25 +ACAACUGGGGCCAGGUG (SEQ 17 upstream ID NO: 606) CEP290-B26 +ACUGGGGCCAGGUGCGG (SEQ 17 upstream ID NO: 607) CEP290-B27 −AUGUGAGCCACCGCACC (SEQ 17 upstream ID NO: 608) CEP290-B31 +AAACUAAGACACUGCCAAUA 20 downstream (SEQ ID NO: 609) CEP290-B32 +AAAACUAAGACACUGCCAAU 20 upstream (SEQ ID NO: 610) CEP290-B33 +AUUACAACUGGGGCCAGGUG 20 upstream (SEQ ID NO: 611) CEP290-B34 +ACAACUGGGGCCAGGUGCGG 20 upstream (SEQ ID NO: 612)

Table 2D provides targeting domains for NHEJ-mediated introduction of anindel in close proximity to or including the LCA10 target position inthe CEP290 gene that can be used for dual targeting. Any of thetargeting domains in the table can be used with a S. pyogenes Cas9(nickase) molecule to generate a single stranded break.

Exemplary nickase pairs including selecting a targeting domain fromGroup A and a second targeting domain from Group B. It is contemplatedherein that a targeting domain of Group A can be combined with any ofthe targeting domains of Group B. For example, the CEP290-B5 orCEP290-B10 can be combined with CEP290-B1 or CEP290-B6.

TABLE 2D Group A Group B CEP290-B5 CEP290-B1 CEP290-B10 CEP290-B6

Table 3A provides targeting domains for NHEJ-mediated introduction of anindel in close proximity to or including the LCA10 target position inthe CEP290 gene selected according to the first tier parameters. Thetargeting domains are within 40 bases of the LCA10 target position, havegood orthogonality, and start with G. It is contemplated herein that thetargeting domain hybridizes to the target domain through complementarybase pairing. Any of the targeting domains in the table can be used witha S. aureus Cas9 molecule that generates a double stranded break (Cas9nuclease) or a single-stranded break (Cas9 nickase).

TABLE 3A Target DNA Site Position relative gRNA Name Strand TargetingDomain Length to mutation CEP290-B1000 + GAGAUACUCACAAUUACAAC 20upstream (SEQ ID NO: 395) CEP290-B1001 + GAUACUCACAAUUACAA 17 upstream(SEQ ID NO: 397)

Table 3B provides targeting domains for NHEJ-mediated introduction of anindel in close proximity to or including the LCA10 target position inthe CEP290 gene selected according to the second tier parameters. Thetargeting domains are within 40 bases of the LCA10 target position, havegood orthogonality, and do not start with G. It is contemplated hereinthat the targeting domain hybridizes to the target domain throughcomplementary base pairing. Any of the targeting domains in the tablecan be used with a S. aureus Cas9 molecule that generates a doublestranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 3B Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B1002 + CACUGCCAAUAGGGAUAGGU 20 downstream(SEQ ID NO: 613) CEP290-B1003 + UGCCAAUAGGGAUAGGU (SEQ 17 downstream IDNO: 614) CEP290-B1004 + UGAGAUACUCACAAUUACAA 20 upstream (SEQ ID NO:615) CEP290-B1005 + AUACUCACAAUUACAAC (SEQ 17 upstream ID NO: 598)

Table 3C provides targeting domains for NHEJ-mediated introduction of anindel in close proximity to or including the LCA10 target position inthe CEP290 gene selected according to the fourth tier parameters. Thetargeting domains are within 40 bases of the LCA10 target position, anddo not start with G. It is contemplated herein that the targeting domainhybridizes to the target domain through complementary base pairing. Anyof the targeting domains in the table can be used with a S. aureus Cas9molecule that generates a double stranded break (Cas9 nuclease) or asingle-stranded break (Cas9 nickase).

TABLE 3C Target DNA Site Position relative gRNA Name Strand TargetingDomain Length to mutation CEP290-B1006 − ACCUGGCCCCAGUUGUAAUU 20upstream (SEQ ID NO: 616) CEP290-B1007 − UGGCCCCAGUUGUAAUU 17 upstream(SEQ ID NO: 617)

Table 4A provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the first tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, have good orthogonality, and start with G.It is contemplated herein that the targeting domain hybridizes to thetarget domain through complementary base pairing. Any of the targetingdomains in the table can be used with a S. pyogenes Cas9 molecule thatgenerates a double stranded break (Cas9 nuclease) or a single-strandedbreak (Cas9 nickase).

TABLE 4A Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B8 − GCUACCGGUUACCUGAA 17 downstream (SEQID NO: 457) CEP290-B217 + GCAGAACUAGUGUAGAC 17 downstream (SEQ ID NO:458) CEP290-B69 − GUUGAGUAUCUCCUGUU 17 downstream (SEQ ID NO: 459)CEP290-B115 + GAUGCAGAACUAGUGUAGAC 20 downstream (SEQ ID NO: 460)CEP290-B187 + GCUUGAACUCUGUGCCAAAC 20 downstream (SEQ ID NO: 461)

Table 4B provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the second tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, have good orthogonality, and do not startwith G. It is contemplated herein that the targeting domain hybridizesto the target domain through complementary base pairing. Any of thetargeting domains in the table can be used with a S. pyogenes Cas9molecule that generates a double stranded break (Cas9 nuclease) or asingle-stranded break (Cas9 nickase).

TABLE 4B Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B269 − AGCUACCGGUUACCUGA 17 downstream(SEQ ID NO: 618) CEP290-B285 + UUUAAGGCGGGGAGUCACAU 20 downstream (SEQID NO: 619) CEP290-B3 − AAAGCUACCGGUUACCUGAA 20 downstream (SEQ ID NO:620) CEP290-B207 − AAAAGCUACCGGUUACCUGA 20 downstream (SEQ ID NO: 621)CEP290-B106 − CUCAUACCUAUCCCUAU (SEQ 17 downstream ID NO: 594)CEP290-B55 + ACACUGCCAAUAGGGAU 17 downstream (SEQ ID NO: 595)CEP290-B138 − UAUCUCAUACCUAUCCCUAU 20 downstream (SEQ ID NO: 599)CEP290-B62 − ACGUGCUCUUUUCUAUAUAU 20 downstream (SEQ ID NO: 622)CEP290-B121 + AUUUGACACCACAUGCACUG 20 downstream (SEQ ID NO: 623)CEP290-B120 − CGUGCUCUUUUCUAUAUAUA 20 downstream (SEQ ID NO: 624)CEP290-B36 − UGGUGUCAAAUAUGGUGCUU 20 downstream (SEQ ID NO: 625)CEP290-B236 + ACUUUUACCCUUCAGGUAAC 20 downstream (SEQ ID NO: 626)CEP290-B70 − AGUGCAUGUGGUGUCAAAUA 20 downstream (SEQ ID NO: 627)CEP290-B177 − UACAUGAGAGUGAUUAGUGG 20 downstream (SEQ ID NO: 628)CEP290-B451 − CGUUGUUCUGAGUAGCUUUC 20 upstream (SEQ ID NO: 629)CEP290-B452 + CCACAAGAUGUCUCUUGCCU 20 upstream (SEQ ID NO: 630)CEP290-B453 − CCUAGGCAAGAGACAUCUUG 20 upstream (SEQ ID NO: 631)CEP290-B454 + UGCCUAGGACUUUCUAAUGC 20 upstream (SEQ ID NO: 632)CEP290-B498 − CGUUGUUCUGAGUAGCUUUC 20 upstream (SEQ ID NO: 629)CEP290-B523 − AUUAGCUCAAAAGCUUUUGC 20 upstream (SEQ ID NO: 633)

Table 4C provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the third tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, and start with G. It is contemplated hereinthat the targeting domain hybridizes to the target domain throughcomplementary base pairing. Any of the targeting domains in the tablecan be used with a S. pyogenes Cas9 molecule that generates a doublestranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 4C Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B87 − GCAUGUGGUGUCAAAUA 17 downstream (SEQID NO: 479) CEP290-B50 + GAUGACAUGAGGUAAGU 17 downstream (SEQ ID NO:478) CEP290-B260 + GUCACAUGGGAGUCACA 17 downstream (SEQ ID NO: 500)CEP290-B283 − GAGAGCCACAGUGCAUG 17 downstream (SEQ ID NO: 472)CEP290-B85 − GCUCUUUUCUAUAUAUA 17 downstream (SEQ ID NO: 481)CEP290-B78 + GCUUUUGACAGUUUUUA 17 downstream (SEQ ID NO: 634)CEP290-B292 + GAUAGAGACAGGAAUAA 17 downstream (SEQ ID NO: 476)CEP290-B278 + GGACUUGACUUUUACCCUUC 20 downstream (SEQ ID NO: 485)CEP290-B227 + GGGAGUCACAUGGGAGUCAC 20 downstream (SEQ ID NO: 491)CEP290-B261 − GUGGAGAGCCACAGUGCAUG 20 downstream (SEQ ID NO: 501)CEP290-B182 + GCCUGAACAAGUUUUGAAAC 20 downstream (SEQ ID NO: 480)CEP290-B67 + GGAGUCACAUGGGAGUCACA 20 downstream (SEQ ID NO: 487)CEP290-B216 + GUAAGACUGGAGAUAGAGAC 20 downstream (SEQ ID NO: 497)CEP290-B241 + GCUUUUGACAGUUUUUAAGG 20 downstream (SEQ ID NO: 482)CEP290-B161 + GUUUAGAAUGAUCAUUCUUG 20 downstream (SEQ ID NO: 504)CEP290-B259 + GUAGCUUUUGACAGUUUUUA 20 downstream (SEQ ID NO: 499)CEP290-B79 + GGAGAUAGAGACAGGAAUAA 20 downstream (SEQ ID NO: 635)CEP290-B436 + GUUCUGUCCUCAGUAAA 17 upstream (SEQ ID NO: 503)CEP290-B444 + GGAUAGGACAGAGGACA 17 upstream (SEQ ID NO: 488)CEP290-B445 + GAUGAAAAAUACUCUUU 17 upstream (SEQ ID NO: 477) CEP290-B459− GAACUCUAUACCUUUUACUG 20 upstream (SEQ ID NO: 466) CEP290-B465 +GUAACAUAAUCACCUCUCUU 20 upstream (SEQ ID NO: 496) CEP290-B473 +GAAAGAUGAAAAAUACUCUU 20 upstream (SEQ ID NO: 462) CEP290-B528 +GUAACAUAAUCACCUCUCUU 20 upstream (SEQ ID NO: 496)

Table 4D provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the fourth tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, and do not start with G. It is contemplatedherein that the targeting domain hybridizes to the target domain throughcomplementary base pairing. Any of the targeting domains in the tablecan be used with a S. pyogenes Cas9 molecule that generates a doublestranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 4D Target DNA Site gRNA Name Strand Targeting Domain LengthCEP290-B233 + AAGGCGGGGAGUCACAU 17 downstream (SEQ ID NO: 636)CEP290-B175 + UAAGGCGGGGAGUCACA 17 downstream (SEQ ID NO: 637)CEP290-B280 + UGAACUCUGUGCCAAAC 17 downstream (SEQ ID NO: 638)CEP290-B92 + CUAAGACACUGCCAAUA 17 downstream (SEQ ID NO: 597)CEP290-B268 + UUUACCCUUCAGGUAAC 17 downstream (SEQ ID NO: 639)CEP290-B154 + UGACACCACAUGCACUG 17 downstream (SEQ ID NO: 640)CEP290-B44 + ACUAAGACACUGCCAAU 17 downstream (SEQ ID NO: 603)CEP290-B231 + UUGCUCUAGAUGACAUG 17 downstream (SEQ ID NO: 641)CEP290-B242 + UGACAGUUUUUAAGGCG 17 downstream (SEQ ID NO: 642)CEP290-B226 − UGUCAAAUAUGGUGCUU 17 downstream (SEQ ID NO: 643)CEP290-B159 + AGUCACAUGGGAGUCAC 17 downstream (SEQ ID NO: 644)CEP290-B222 − AUGAGAGUGAUUAGUGG 17 downstream (SEQ ID NO: 645)CEP290-B274 + UGACAUGAGGUAAGUAG 17 downstream (SEQ ID NO: 646)CEP290-B68 − UACAUGAGAGUGAUUAG 17 downstream (SEQ ID NO: 647)CEP290-B212 + UAAGGAGGAUGUAAGAC 17 downstream (SEQ ID NO: 648)CEP290-B270 + CUUGACUUUUACCCUUC 17 downstream (SEQ ID NO: 649)CEP290-B96 + UCACUGAGCAAAACAAC 17 downstream (SEQ ID NO: 650)CEP290-B104 + AGACUUAUAUUCCAUUA 17 downstream (SEQ ID NO: 651)CEP290-B122 + CAUGGGAGUCACAGGGU 17 downstream (SEQ ID NO: 652)CEP290-B229 + UAGAAUGAUCAUUCUUG 17 downstream (SEQ ID NO: 653)CEP290-B99 + UUGACAGUUUUUAAGGC 17 downstream (SEQ ID NO: 654) CEP290-B7− AAACUGUCAAAAGCUAC 17 downstream (SEQ ID NO: 655) CEP290-B41 +UCAUUCUUGUGGCAGUA 17 downstream (SEQ ID NO: 2780) CEP290-B37 +AUGACAUGAGGUAAGUA 17 downstream (SEQ ID NO: 656) CEP290-B97 −UGUUUCAAAACUUGUUC 17 downstream (SEQ ID NO: 657) CEP290-B173 −AUAUCUGUCUUCCUUAA 17 downstream (SEQ ID NO: 658) CEP290-B136 +UGAACAAGUUUUGAAAC 17 downstream (SEQ ID NO: 659) CEP290-B71 −UUCUGCAUCUUAUACAU 17 downstream (SEQ ID NO: 660) CEP290-B172 −AUAAGUCUUUUGAUAUA 17 downstream (SEQ ID NO: 661) CEP290-B238 +UUUGACAGUUUUUAAGG 17 downstream (SEQ ID NO: 662) CEP290-B148 −UGCUCUUUUCUAUAUAU 17 downstream (SEQ ID NO: 663) CEP290-B208 +AGACUGGAGAUAGAGAC 17 downstream (SEQ ID NO: 664) CEP290-B53 +CAUAAGAAAGAACACUG 17 downstream (SEQ ID NO: 665) CEP290-B166 +UUCUUGUGGCAGUAAGG 17 downstream (SEQ ID NO: 666) CEP290-B247 −AAGCAUACUUUUUUUAA 17 downstream (SEQ ID NO: 667) CEP290-B245 +CAACUGGAAGAGAGAAA 17 downstream (SEQ ID NO: 668) CEP290-B167 +UAUGCUUAAGAAAAAAA 17 downstream (SEQ ID NO: 669) CEP290-B171 −UUUUAUUAUCUUUAUUG 17 downstream (SEQ ID NO: 670) CEP290-B140 +CUAGAUGACAUGAGGUAAGU 20 downstream (SEQ ID NO: 671) CEP290-B147 +UUUUAAGGCGGGGAGUCACA 20 downstream (SEQ ID NO: 672) CEP290-B253 +AAGACACUGCCAAUAGGGAU 20 downstream (SEQ ID NO: 600) CEP290-B73 −UCCUGUUUCAAAACUUGUUC 20 downstream (SEQ ID NO: 673) CEP290-B206 −UGUGUUGAGUAUCUCCUGUU 20 downstream (SEQ ID NO: 674) CEP290-B57 +CUCUUGCUCUAGAUGACAUG 20 downstream (SEQ ID NO: 675) CEP290-B82 +CAGUAAGGAGGAUGUAAGAC 20 downstream (SEQ ID NO: 676) CEP290-B265 +AGAUGACAUGAGGUAAGUAG 20 downstream (SEQ ID NO: 677) CEP290-B105 +AAUUCACUGAGCAAAACAAC 20 downstream (SEQ ID NO: 678) CEP290-B239 +UCACAUGGGAGUCACAGGGU 20 downstream (SEQ ID NO: 679) CEP290-B180 +UAGAUGACAUGAGGUAAGUA 20 downstream (SEQ ID NO: 680) CEP290-B103 +UUUUGACAGUUUUUAAGGCG 20 downstream (SEQ ID NO: 681) CEP290-B254 −UAAUACAUGAGAGUGAUUAG 20 downstream (SEQ ID NO: 682) CEP290-B134 −UAGUUCUGCAUCUUAUACAU 20 downstream (SEQ ID NO: 683) CEP290-B151 +AAACUAAGACACUGCCAAUA 20 downstream (SEQ ID NO: 609) CEP290-B196 +AAAACUAAGACACUGCCAAU 20 downstream (SEQ ID NO: 610) CEP290-B2 −UAAAAACUGUCAAAAGCUAC 20 downstream (SEQ ID NO: 506) CEP290-B240 +CUUUUGACAGUUUUUAAGGC 20 downstream (SEQ ID NO: 684) CEP290-B116 +AAAAGACUUAUAUUCCAUUA 20 downstream (SEQ ID NO: 685) CEP290-B39 +AUACAUAAGAAAGAACACUG 20 downstream (SEQ ID NO: 686) CEP290-B91 −AAUAUAAGUCUUUUGAUAUA 20 downstream (SEQ ID NO: 687) CEP290-B126 +UGAUCAUUCUUGUGGCAGUA 20 downstream (SEQ ID NO: 688) CEP290-B202 −UACAUAUCUGUCUUCCUUAA 20 downstream (SEQ ID NO: 689) CEP290-B152 −CUUAAGCAUACUUUUUUUAA 20 downstream (SEQ ID NO: 690) CEP290-B77 +AAACAACUGGAAGAGAGAAA 20 downstream (SEQ ID NO: 691) CEP290-B145 +UCAUUCUUGUGGCAGUAAGG 20 downstream (SEQ ID NO: 692) CEP290-B72 +AAGUAUGCUUAAGAAAAAAA 20 downstream (SEQ ID NO: 693) CEP290-B221 −AUUUUUUAUUAUCUUUAUUG 20 downstream (SEQ ID NO: 694) CEP290-B424 +CUAGGACUUUCUAAUGC 17 upstream (SEQ ID NO: 695) CEP290-B425 −AUCUAAGAUCCUUUCAC 17 upstream (SEQ ID NO: 696) CEP290-B426 +UUAUCACCACACUAAAU 17 upstream (SEQ ID NO: 697) CEP290-B427 −AGCUCAAAAGCUUUUGC 17 upstream (SEQ ID NO: 698) CEP290-B428 −UGUUCUGAGUAGCUUUC 17 upstream (SEQ ID NO: 699) CEP290-B429 +ACUUUCUAAUGCUGGAG 17 upstream (SEQ ID NO: 700) CEP290-B430 −CUCUAUACCUUUUACUG 17 upstream (SEQ ID NO: 701) CEP290-B431 +CAAGAUGUCUCUUGCCU 17 upstream (SEQ ID NO: 702) CEP290-B432 −AUUAUGCCUAUUUAGUG 17 upstream (SEQ ID NO: 703) CEP290-B433 +AUGACUCAUAAUUUAGU 17 upstream (SEQ ID NO: 704) CEP290-B434 −UAGAGGCUUAUGGAUUU 17 upstream (SEQ ID NO: 705) CEP290-B435 +UAUUCUACUCCUGUGAA 17 upstream (SEQ ID NO: 706) CEP290-B437 +CUAAUGCUGGAGAGGAU 17 upstream (SEQ ID NO: 707) CEP290-B438 −AGGCAAGAGACAUCUUG 17 upstream (SEQ ID NO: 708) CEP290-B439 +AGCCUCUAUUUCUGAUG 17 upstream (SEQ ID NO: 709) CEP290-B440 −CAGCAUUAGAAAGUCCU 17 upstream (SEQ ID NO: 710) CEP290-B441 −CUGCUUUUGCCAAAGAG 17 upstream (SEQ ID NO: 711) CEP290-B442 +ACAUAAUCACCUCUCUU 17 upstream (SEQ ID NO: 712) CEP290-B443 −UCAGAAAUAGAGGCUUA 17 upstream (SEQ ID NO: 713) CEP290-B446 −UUCCUCAUCAGAAAUAG 17 upstream (SEQ ID NO: 714) CEP290-B447 +ACAGAGGACAUGGAGAA 17 upstream (SEQ ID NO: 715) CEP290-B448 +UGGAGAGGAUAGGACAG 17 upstream (SEQ ID NO: 716) CEP290-B449 +AGGAAGAUGAACAAAUC 17 upstream (SEQ ID NO: 717) CEP290-B450 +AGAUGAAAAAUACUCUU 17 upstream (SEQ ID NO: 718) CEP290-B455 +AGGACUUUCUAAUGCUGGAG 20 upstream (SEQ ID NO: 719) CEP290-B456 −AUUAGCUCAAAAGCUUUUGC 20 upstream (SEQ ID NO: 633) CEP290-B457 −CUCCAGCAUUAGAAAGUCCU 20 upstream (SEQ ID NO: 720) CEP290-B458 +AACAUGACUCAUAAUUUAGU 20 upstream (SEQ ID NO: 721) CEP290-B460 −AUCUUCCUCAUCAGAAAUAG 20 upstream (SEQ ID NO: 722) CEP290-B461 +AUAAGCCUCUAUUUCUGAUG 20 upstream (SEQ ID NO: 723) CEP290-B462 +UCUUAUUCUACUCCUGUGAA 20 upstream (SEQ ID NO: 724) CEP290-B463 −CUGCUGCUUUUGCCAAAGAG 20 upstream (SEQ ID NO: 725) CEP290-B464 +UUUCUAAUGCUGGAGAGGAU 20 upstream (SEQ ID NO: 726) CEP290-B466 +AAAUUAUCACCACACUAAAU 20 upstream (SEQ ID NO: 727) CEP290-B467 +CUUGUUCUGUCCUCAGUAAA 20 upstream (SEQ ID NO: 728) CEP290-B468 −AAAAUUAUGCCUAUUUAGUG 20 upstream (SEQ ID NO: 729) CEP290-B469 −UCAUCAGAAAUAGAGGCUUA 20 upstream (SEQ ID NO: 730) CEP290-B470 −AAAUAGAGGCUUAUGGAUUU 20 upstream (SEQ ID NO: 731) CEP290-B471 +UGCUGGAGAGGAUAGGACAG 20 upstream (SEQ ID NO: 732) CEP290-B472 +AUGAGGAAGAUGAACAAAUC 20 upstream (SEQ ID NO: 733) CEP290-B474 −CUUAUCUAAGAUCCUUUCAC 20 upstream (SEQ ID NO: 734) CEP290-B475 +AGAGGAUAGGACAGAGGACA 20 upstream (SEQ ID NO: 735) CEP290-B476 +AGGACAGAGGACAUGGAGAA 20 upstream (SEQ ID NO: 736) CEP290-B477 +AAAGAUGAAAAAUACUCUUU 20 upstream (SEQ ID NO: 737) CEP290-B495 −AGCUCAAAAGCUUUUGC 17 upstream (SEQ ID NO: 698) CEP290-B529 −UGUUCUGAGUAGCUUUC 17 upstream (SEQ ID NO: 699) CEP290-B513 +AUGACUCAUAAUUUAGU 17 upstream (SEQ ID NO: 704) CEP290-B490 +UAUUCUACUCCUGUGAA 17 upstream (SEQ ID NO: 706) CEP290-B485 −CUGCUUUUGCCAAAGAG 17 upstream (SEQ ID NO: 711) CEP290-B492 +ACAUAAUCACCUCUCUU 17 upstream (SEQ ID NO: 712) CEP290-B506 +AACAUGACUCAUAAUUUAGU 20 upstream (SEQ ID NO: 721) CEP290-B500 +UCUUAUUCUACUCCUGUGAA 20 upstream (SEQ ID NO: 724) CEP290-B521 −CUGCUGCUUUUGCCAAAGAG 20 upstream (SEQ ID NO: 725)

Table 5A provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the first tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, have good orthogonality, and start with G.It is contemplated herein that the targeting domain hybridizes to thetarget domain through complementary base pairing. Any of the targetingdomains in the table can be used with a S. aureus Cas9 molecule thatgenerates a double stranded break (Cas9 nuclease) or a single-strandedbreak (Cas9 nickase).

TABLE 5A Position Target relative DNA Site to gRNA Name Strand TargetingDomain Length mutation CEP290- + GAAUCCUGAAAGCUACU 17 upstream B1008(SEQ ID NO: 510)

Table 5B provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the second tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, have good orthogonality, and do not startwith G. It is contemplated herein that the targeting domain hybridizesto the target domain through complementary base pairing. Any of thetargeting domains in the table can be used with a S. aureus Cas9molecule that generates a double stranded break (Cas9 nuclease) or asingle-stranded break (Cas9 nickase).

TABLE 5B Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B1009 − CCUACUUACCUCAUGUCAUC 20 downstream(SEQ ID NO: 747) CEP290-B1010 + CUAUGAGCCAGCAAAAGCUU 20 upstream (SEQ IDNO: 748) CEP290-B1011 − ACGUUGUUCUGAGUAGCUUU 20 upstream (SEQ ID NO:749) CEP290-B1012 − CAUAGAGACACAUUCAGUAA 20 upstream (SEQ ID NO: 750)CEP290-B1013 − ACUUACCUCAUGUCAUC 17 downstream (SEQ ID NO: 751)CEP290-B1014 + UGAGCCAGCAAAAGCUU 17 upstream (SEQ ID NO: 752)CEP290-B1015 − UUGUUCUGAGUAGCUUU 17 upstream (SEQ ID NO: 753)CEP290-B1016 − AGAGACACAUUCAGUAA 17 upstream (SEQ ID NO: 754)CEP290-B1017 + UUUAAGGCGGGGAGUCACAU 20 downstream (SEQ ID NO: 619)CEP290-B1018 − CAAAAGCUACCGGUUACCUG 20 downstream (SEQ ID NO: 755)CEP290-B1019 + UUUUAAGGCGGGGAGUCACA 20 downstream (SEQ ID NO: 672)CEP290-B1020 − UGUCAAAAGCUACCGGUUAC 20 downstream (SEQ ID NO: 757)CEP290-B1021 + AAGGCGGGGAGUCACAU 17 downstream (SEQ ID NO: 636)CEP290-B1022 − AAGCUACCGGUUACCUG 17 downstream (SEQ ID NO: 758)CEP290-B1023 + UAAGGCGGGGAGUCACA 17 downstream (SEQ ID NO: 637)CEP290-B1024 − CAAAAGCUACCGGUUAC 17 downstream (SEQ ID NO: 759)CEP290-B1025 + UAGGAAUCCUGAAAGCUACU 20 upstream (SEQ ID NO: 760)CEP290-B1026 + CAGAACAACGUUUUCAUUUA 20 upstream (SEQ ID NO: 761)CEP290-B1027 − CAAAAGCUUUUGCUGGCUCA 20 upstream (SEQ ID NO: 762)CEP290-B1028 + AGCAAAAGCUUUUGAGCUAA 20 upstream (SEQ ID NO: 763)CEP290-B1029 + AUCUUAUUCUACUCCUGUGA 20 upstream (SEQ ID NO: 764)CEP290-B1030 + AACAACGUUUUCAUUUA 17 upstream (SEQ ID NO: 765)CEP290-B1031 − AAGCUUUUGCUGGCUCA 17 upstream (SEQ ID NO: 766)CEP290-B1032 + AAAAGCUUUUGAGCUAA 17 upstream (SEQ ID NO: 767)CEP290-B1033 + UUAUUCUACUCCUGUGA 17 upstream (SEQ ID NO: 768)

Table 5C provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the third tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, and start with G. It is contemplated hereinthat the targeting domain hybridizes to the target domain throughcomplementary base pairing. Any of the targeting domains in the tablecan be used with a S. aureus Cas9 molecule that generates a doublestranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 5C Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B1034 + GAAACAGGAAUAGAAAUUCA 20 downstream(SEQ ID NO: 769) CEP290-B1035 + GAAAGAUGAAAAAUACUCUU 20 upstream (SEQ IDNO: 462) CEP290-B1036 − GAAAUAGAGGCUUAUGGAUU 20 upstream (SEQ ID NO:547) CEP290-B1037 − GAAUAUAAGUCUUUUGAUAU 20 downstream (SEQ ID NO: 770)CEP290-B1038 + GAGAAAUGGUUCCCUAUAUA 20 downstream (SEQ ID NO: 771)CEP290-B1039 + GAGAGGAUAGGACAGAGGAC 20 upstream (SEQ ID NO: 772)CEP290-B1040 + GAUGAGGAAGAUGAACAAAU 20 upstream (SEQ ID NO: 773)CEP290-B1041 + GAUGCAGAACUAGUGUAGAC 20 downstream (SEQ ID NO: 460)CEP290-B1042 − GAUUUGUUCAUCUUCCUCAU 20 upstream (SEQ ID NO: 774)CEP290-B1043 + GCAGUAAGGAGGAUGUAAGA 20 downstream (SEQ ID NO: 775)CEP290-B1044 + GCCUGAACAAGUUUUGAAAC 20 downstream (SEQ ID NO: 480)CEP290-B1045 + GCUUGAACUCUGUGCCAAAC 20 downstream (SEQ ID NO: 461)CEP290-B1046 − GCUUUCUGCUGCUUUUGCCA 20 upstream (SEQ ID NO: 776)CEP290-B1047 − GCUUUCUGCUGCUUUUGCCA 20 upstream (SEQ ID NO: 776)CEP290-B1048 + GCUUUUGACAGUUUUUAAGG 20 downstream (SEQ ID NO: 482)CEP290-B1049 + GGAAAGAUGAAAAAUACUCU 20 upstream (SEQ ID NO: 778)CEP290-B1050 + GGAGGAUGUAAGACUGGAGA 20 downstream (SEQ ID NO: 779)CEP290-B1051 + GGGGAGUCACAUGGGAGUCA 20 downstream (SEQ ID NO: 573)CEP290-B1052 − GGUGAUUAUGUUACUUUUUA 20 upstream (SEQ ID NO: 780)CEP290-B1053 − GGUGAUUAUGUUACUUUUUA 20 upstream (SEQ ID NO: 780)CEP290-B1054 + GUAAGACUGGAGAUAGAGAC 20 downstream (SEQ ID NO: 497)CEP290-B1055 + GUCACAUGGGAGUCACAGGG 20 downstream (SEQ ID NO: 586)CEP290-B1056 − GUGGUGUCAAAUAUGGUGCU 20 downstream (SEQ ID NO: 782)CEP290-B1057 + GAAAAAAAAGGUAAUGC 17 downstream (SEQ ID NO: 783)CEP290-B1058 + GAAAAGAGCACGUACAA 17 downstream (SEQ ID NO: 784CEP290-B1059 + GAAUCCUGAAAGCUACU 17 upstream (SEQ ID NO: 510)CEP290-B1060 − GAAUGAUCAUUCUAAAC 17 downstream (SEQ ID NO: 785)CEP290-B1061 + GACAGAGGACAUGGAGA 17 upstream (SEQ ID NO: 786)CEP290-B1062 + GACUUUCUAAUGCUGGA 17 upstream (SEQ ID NO: 787)CEP290-B1063 − GAGAGUGAUUAGUGGUG 17 downstream (SEQ ID NO: 788)CEP290-B1064 + GAGCAAAACAACUGGAA 17 downstream (SEQ ID NO: 789)CEP290-B1065 + GAGGAAGAUGAACAAAU 17 upstream (SEQ ID NO: 790)CEP290-B1066 + GAGUCACAUGGGAGUCA 17 downstream (SEQ ID NO: 791)CEP290-B1067 + GAUCUUAUUCUACUCCU 17 upstream (SEQ ID NO: 792)CEP290-B1068 + GAUCUUAUUCUACUCCU 17 upstream (SEQ ID NO: 792)CEP290-B1069 + GAUGAAAAAUACUCUUU 17 upstream (SEQ ID NO: 477)CEP290-B1070 + GAUGACAUGAGGUAAGU 17 downstream (SEQ ID NO: 478)CEP290-B1071 − GAUUAUGUUACUUUUUA 17 upstream (SEQ ID NO: 793)CEP290-B1072 − GAUUAUGUUACUUUUUA 17 upstream (SEQ ID NO: 793)CEP290-B1073 + GCAAAACAACUGGAAGA 17 downstream (SEQ ID NO: 794)CEP290-B1074 + GCAGAACUAGUGUAGAC 17 downstream (SEQ ID NO: 458)CEP290-B1075 − GCUCUUUUCUAUAUAUA 17 downstream (SEQ ID NO: 481)CEP290-B1076 + GGAUAGGACAGAGGACA 17 upstream (SEQ ID NO: 488)CEP290-B1077 + GGAUGUAAGACUGGAGA 17 downstream (SEQ ID NO: 795)CEP290-B1078 + GUAAGGAGGAUGUAAGA 17 downstream (SEQ ID NO: 796)CEP290-B1079 − GUAUCUCCUGUUUGGCA 17 downstream (SEQ ID NO: 797)CEP290-B1080 − GUCAUCUAGAGCAAGAG 17 downstream (SEQ ID NO: 798)CEP290-B1081 + GUCCUCAGUAAAAGGUA 17 upstream (SEQ ID NO: 799)CEP290-B1082 + GUGAAAGGAUCUUAGAU 17 upstream (SEQ ID NO: 800)CEP290-B1083 − GUGCUCUUUUCUAUAUA 17 downstream (SEQ ID NO: 801)CEP290-B1084 − GUGUCAAAUAUGGUGCU 17 downstream (SEQ ID NO: 802)CEP290-B1085 + GUUCCCUAUAUAUAGAA 17 downstream (SEQ ID NO: 803)

Table 5D provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the fourth tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, and do not start with G. It is contemplatedherein that the targeting domain hybridizes to the target domain throughcomplementary base pairing. Any of the targeting domains in the tablecan be used with a S. aureus Cas9 molecule that generates a doublestranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 5D Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B1086 + AAAACUAAGACACUGCCAAU 20 downstream(SEQ ID NO: 610) CEP290-B1087 + AAAAGACUUAUAUUCCAUUA 20 downstream (SEQID NO: 685) CEP290-B1088 + AAACAUGACUCAUAAUUUAG 20 upstream (SEQ ID NO:805) CEP290-B1089 + AAACAUGACUCAUAAUUUAG 20 upstream (SEQ ID NO: 805)CEP290-B1090 + AAAGAUGAAAAAUACUCUUU 20 upstream (SEQ ID NO: 737)CEP290-B1091 + AAAUUCACUGAGCAAAACAA 20 downstream (SEQ ID NO: 808)CEP290-B1092 + AACAAGUUUUGAAACAGGAA 20 downstream (SEQ ID NO: 809)CEP290-B1093 + AACAGGAGAUACUCAACACA 20 downstream (SEQ ID NO: 810)CEP290-B1094 + AACAUGACUCAUAAUUUAGU 20 upstream (SEQ ID NO: 721)CEP290-B1095 + AACAUGACUCAUAAUUUAGU 20 upstream (SEQ ID NO: 721)CEP290-B1096 − AAUAUAAGUCUUUUGAUAUA 20 downstream (SEQ ID NO: 687)CEP290-B1097 + AAUCACUCUCAUGUAUUAGC 20 downstream (SEQ ID NO: 814)CEP290-B1098 + AAUUCACUGAGCAAAACAAC 20 downstream (SEQ ID NO: 678)CEP290-B1099 + ACAAAAGAACAUACAUAAGA 20 downstream (SEQ ID NO: 816)CEP290-B1100 + ACGUACAAAAGAACAUACAU 20 downstream (SEQ ID NO: 817)CEP290-B1101 − ACGUGCUCUUUUCUAUAUAU 20 downstream (SEQ ID NO: 622)CEP290-B1102 − ACGUUGUUCUGAGUAGCUUU 20 upstream (SEQ ID NO: 749)CEP290-B1103 + ACUGAGCAAAACAACUGGAA 20 downstream (SEQ ID NO: 819)CEP290-B1104 + AGAGGAUAGGACAGAGGACA 20 upstream (SEQ ID NO: 735)CEP290-B1105 + AGAUGCAGAACUAGUGUAGA 20 downstream (SEQ ID NO: 821)CEP290-B1106 + AGCAAAAGCUUUUGAGCUAA 20 upstream (SEQ ID NO: 763)CEP290-B1107 − AGCAUUAGAAAGUCCUAGGC 20 upstream (SEQ ID NO: 823)CEP290-B1108 + AGCUUGAACUCUGUGCCAAA 20 downstream (SEQ ID NO: 824)CEP290-B1109 + AGCUUUUGACAGUUUUUAAG 20 downstream (SEQ ID NO: 825)CEP290-B1110 + AGGACAGAGGACAUGGAGAA 20 upstream (SEQ ID NO: 736)CEP290-B1111 + AGGAUAGGACAGAGGACAUG 20 upstream (SEQ ID NO: 827)CEP290-B1112 + AGGUAAUGCCUGAACAAGUU 20 downstream (SEQ ID NO: 828)CEP290-B1113 + AUAAGAAAGAACACUGUGGU 20 downstream (SEQ ID NO: 829)CEP290-B1114 + AUAAGCCUCUAUUUCUGAUG 20 upstream (SEQ ID NO: 723)CEP290-B1115 − AUACAUGAGAGUGAUUAGUG 20 downstream (SEQ ID NO: 831)CEP290-B1116 + AUAGAAAAGAGCACGUACAA 20 downstream (SEQ ID NO: 832)CEP290-B1117 + AUCAUUCUUGUGGCAGUAAG 20 downstream (SEQ ID NO: 833)CEP290-B1118 + AUCUUAUUCUACUCCUGUGA 20 upstream (SEQ ID NO: 764)CEP290-B1119 − AUCUUGUGGAUAAUGUAUCA 20 upstream (SEQ ID NO: 835)CEP290-B1120 + AUGAGGAAGAUGAACAAAUC 20 upstream (SEQ ID NO: 733)CEP290-B1121 + AUGAUCAUUCUUGUGGCAGU 20 downstream (SEQ ID NO: 837)CEP290-B1122 + AUGCUGGAGAGGAUAGGACA 20 upstream (SEQ ID NO: 838)CEP290-B1123 + AUGGUUCCCUAUAUAUAGAA 20 downstream (SEQ ID NO: 839)CEP290-B1124 − AUUUAAUUUGUUUCUGUGUG 20 downstream (SEQ ID NO: 840)CEP290-B1125 + CAAAACCUAUGUAUAAGAUG 20 downstream (SEQ ID NO: 841)CEP290-B1126 + CAAAAGACUUAUAUUCCAUU 20 downstream (SEQ ID NO: 842)CEP290-B1127 − CAAAAGCUUUUGCUGGCUCA 20 upstream (SEQ ID NO: 762)CEP290-B1128 − CAAGAAUGAUCAUUCUAAAC 20 downstream (SEQ ID NO: 844)CEP290-B1129 − CACAGAGUUCAAGCUAAUAC 20 downstream (SEQ ID NO: 845)CEP290-B1130 + CACAGGGUAGGAUUCAUGUU 20 downstream (SEQ ID NO: 846)CEP290-B1131 + CACUGCCAAUAGGGAUAGGU 20 downstream (SEQ ID NO: 613)CEP290-B1132 + CAGAACAACGUUUUCAUUUA 20 upstream (SEQ ID NO: 761)CEP290-B1133 − CAGAGUUCAAGCUAAUACAU 20 downstream (SEQ ID NO: 848)CEP290-B1134 − CAGUAAAUGAAAACGUUGUU 20 upstream (SEQ ID NO: 849)CEP290-B1135 − CAGUAAAUGAAAACGUUGUU 20 upstream (SEQ ID NO: 849)CEP290-B1136 + CAGUAAGGAGGAUGUAAGAC 20 downstream (SEQ ID NO: 676)CEP290-B1137 + CAUAAGCCUCUAUUUCUGAU 20 upstream (SEQ ID NO: 851)CEP290-B1138 − CAUAGAGACACAUUCAGUAA 20 upstream (SEQ ID NO: 750)CEP290-B1139 + CAUCUCUUGCUCUAGAUGAC 20 downstream (SEQ ID NO: 853)CEP290-B1140 − CAUGAGAGUGAUUAGUGGUG 20 downstream (SEQ ID NO: 854)CEP290-B1141 − CAUGUCAUCUAGAGCAAGAG 20 downstream (SEQ ID NO: 855)CEP290-B1142 + CAUUUACUGAAUGUGUCUCU 20 upstream (SEQ ID NO: 856)CEP290-B1143 + CAUUUACUGAAUGUGUCUCU 20 upstream (SEQ ID NO: 856)CEP290-B1144 + CCAUUAAAAAAAGUAUGCUU 20 downstream (SEQ ID NO: 857)CEP290-B1145 + CCUAGGACUUUCUAAUGCUG 20 upstream (SEQ ID NO: 858)CEP290-B1146 + CCUCUCUUUGGCAAAAGCAG 20 upstream (SEQ ID NO: 859)CEP290-B1147 + CCUCUCUUUGGCAAAAGCAG 20 upstream (SEQ ID NO: 859)CEP290-B1148 + CCUGUGAAAGGAUCUUAGAU 20 upstream (SEQ ID NO: 860)CEP290-B1149 − CGUGCUCUUUUCUAUAUAUA 20 downstream (SEQ ID NO: 624)CEP290-B1150 − CUAAGAUCCUUUCACAGGAG 20 upstream (SEQ ID NO: 861)CEP290-B1151 + CUAGAUGACAUGAGGUAAGU 20 downstream (SEQ ID NO: 671)CEP290-B1152 + CUAUGAGCCAGCAAAAGCUU 20 upstream (SEQ ID NO: 748)CEP290-B1153 + CUCAUAAUUUAGUAGGAAUC 20 upstream (SEQ ID NO: 864)CEP290-B1154 + CUCAUAAUUUAGUAGGAAUC 20 upstream (SEQ ID NO: 864)CEP290-B1155 − CUCAUCAGAAAUAGAGGCUU 20 upstream (SEQ ID NO: 865)CEP290-B1156 + CUCUAUUUCUGAUGAGGAAG 20 upstream (SEQ ID NO: 866)CEP290-B1157 − CUUAAGCAUACUUUUUUUAA 20 downstream (SEQ ID NO: 690)CEP290-B1158 − CUUAUCUAAGAUCCUUUCAC 20 upstream (SEQ ID NO: 734)CEP290-B1159 + CUUUCUAAUGCUGGAGAGGA 20 upstream (SEQ ID NO: 869)CEP290-B1160 + CUUUUGACAGUUUUUAAGGC 20 downstream (SEQ ID NO: 684)CEP290-B1161 + UAAAACUAAGACACUGCCAA 20 downstream (SEQ ID NO: 871)CEP290-B1162 + UAAGAAAAAAAAGGUAAUGC 20 downstream (SEQ ID NO: 872)CEP290-B1163 + UAAUGCUGGAGAGGAUAGGA 20 upstream (SEQ ID NO: 873)CEP290-B1164 − UACAUAUCUGUCUUCCUUAA 20 downstream (SEQ ID NO: 689)CEP290-B1165 − UACAUCCUCCUUACUGCCAC 20 downstream (SEQ ID NO: 875)CEP290-B1166 − UACAUGAGAGUGAUUAGUGG 20 downstream (SEQ ID NO: 628)CEP290-B1167 − UACCUCAUGUCAUCUAGAGC 20 downstream (SEQ ID NO: 876)CEP290-B1168 − UACGUGCUCUUUUCUAUAUA 20 downstream (SEQ ID NO: 877)CEP290-B1169 − UAGAGCAAGAGAUGAACUAG 20 downstream (SEQ ID NO: 878)CEP290-B1170 + UAGAUGACAUGAGGUAAGUA 20 downstream (SEQ ID NO: 680)CEP290-B1171 + UAGGAAUCCUGAAAGCUACU 20 upstream (SEQ ID NO: 760)CEP290-B1172 + UAGGACAGAGGACAUGGAGA 20 upstream (SEQ ID NO: 881)CEP290-B1173 + UAGGACUUUCUAAUGCUGGA 20 upstream (SEQ ID NO: 882)CEP290-B1174 + UCACUGAGCAAAACAACUGG 20 downstream (SEQ ID NO: 883)CEP290-B1175 − UCAUGUUUAUCAAUAUUAUU 20 upstream (SEQ ID NO: 884)CEP290-B1176 − UCAUGUUUAUCAAUAUUAUU 20 upstream (SEQ ID NO: 884)CEP290-B1177 + UCCACAAGAUGUCUCUUGCC 20 upstream (SEQ ID NO: 885)CEP290-B1178 + UCCAUAAGCCUCUAUUUCUG 20 upstream (SEQ ID NO: 886)CEP290-B1179 − UCCUAGGCAAGAGACAUCUU 20 upstream (SEQ ID NO: 887)CEP290-B1180 + UCUAGAUGACAUGAGGUAAG 20 downstream (SEQ ID NO: 888)CEP290-B1181 − UCUAUACCUUUUACUGAGGA 20 upstream (SEQ ID NO: 889)CEP290-B1182 + UCUGUCCUCAGUAAAAGGUA 20 upstream (SEQ ID NO: 890)CEP290-B1183 − UCUUAAGCAUACUUUUUUUA 20 downstream (SEQ ID NO: 891)CEP290-B1184 − UCUUAUCUAAGAUCCUUUCA 20 upstream (SEQ ID NO: 892)CEP290-B1185 − UCUUCCAGUUGUUUUGCUCA 20 downstream (SEQ ID NO: 893)CEP290-B1186 + UGAGCAAAACAACUGGAAGA 20 downstream (SEQ ID NO: 894)CEP290-B1187 − UGAGUAUCUCCUGUUUGGCA 20 downstream (SEQ ID NO: 895)CEP290-B1188 + UGAUCAUUCUUGUGGCAGUA 20 downstream (SEQ ID NO: 688)CEP290-B1189 + UGCCUAGGACUUUCUAAUGC 20 upstream (SEQ ID NO: 632)CEP290-B1190 + UGCCUGAACAAGUUUUGAAA 20 downstream (SEQ ID NO: 897)CEP290-B1191 − UGGUGUCAAAUAUGGUGCUU 20 downstream (SEQ ID NO: 625)CEP290-B1192 + UGUAAGACUGGAGAUAGAGA 20 downstream (SEQ ID NO: 898)CEP290-B1193 − UGUCCUAUCCUCUCCAGCAU 20 upstream (SEQ ID NO: 899)CEP290-B1194 − UUAACGUUAUCAUUUUCCCA 20 upstream (SEQ ID NO: 900)CEP290-B1195 − UUACAUAUCUGUCUUCCUUA 20 downstream (SEQ ID NO: 901)CEP290-B1196 + UUAGAUCUUAUUCUACUCCU 20 upstream (SEQ ID NO: 902)CEP290-B1197 + UUAGAUCUUAUUCUACUCCU 20 upstream (SEQ ID NO: 902)CEP290-B1198 − UUCAGGAUUCCUACUAAAUU 20 upstream (SEQ ID NO: 904)CEP290-B1199 − UUCAGGAUUCCUACUAAAUU 20 upstream (SEQ ID NO: 904)CEP290-B1200 − UUCAUCUUCCUCAUCAGAAA 20 upstream (SEQ ID NO: 905)CEP290-B1201 + UUGCCUAGGACUUUCUAAUG 20 upstream (SEQ ID NO: 906)CEP290-B1202 − UUUCUGCUGCUUUUGCCAAA 20 upstream (SEQ ID NO: 907)CEP290-B1203 − UUUCUGCUGCUUUUGCCAAA 20 upstream (SEQ ID NO: 907)CEP290-B1204 + UUUUGACAGUUUUUAAGGCG 20 downstream (SEQ ID NO: 681)CEP290-B1205 + UUUUUAAGGCGGGGAGUCAC 20 downstream (SEQ ID NO: 909)CEP290-B1206 + AAAAGCUUUUGAGCUAA 17 upstream (SEQ ID NO: 767)CEP290-B1207 + AAAGAACAUACAUAAGA 17 downstream (SEQ ID NO: 911)CEP290-B1208 + AAAUGGUUCCCUAUAUA 17 downstream (SEQ ID NO: 912)CEP290-B1209 + AACAACGUUUUCAUUUA 17 upstream (SEQ ID NO: 765)CEP290-B1210 + AACCUAUGUAUAAGAUG 17 downstream (SEQ ID NO: 914)CEP290-B1211 + AACUAAGACACUGCCAA 17 downstream (SEQ ID NO: 915)CEP290-B1212 + AAGACUGGAGAUAGAGA 17 downstream (SEQ ID NO: 916)CEP290-B1213 + AAGACUUAUAUUCCAUU 17 downstream (SEQ ID NO: 917)CEP290-B1214 + AAGAUGAAAAAUACUCU 17 upstream (SEQ ID NO: 918)CEP290-B1215 − AAGCAUACUUUUUUUAA 17 downstream (SEQ ID NO: 667)CEP290-B1216 + AAGCCUCUAUUUCUGAU 17 upstream (SEQ ID NO: 920)CEP290-B1217 − AAGCUUUUGCUGGCUCA 17 upstream (SEQ ID NO: 766)CEP290-B1218 + AAGUUUUGAAACAGGAA 17 downstream (SEQ ID NO: 922)CEP290-B1219 + ACAAGAUGUCUCUUGCC 17 upstream (SEQ ID NO: 923)CEP290-B1220 + ACAGAGGACAUGGAGAA 17 upstream (SEQ ID NO: 715)CEP290-B1221 + ACAGGAAUAGAAAUUCA 17 downstream (SEQ ID NO: 925)CEP290-B1222 + ACAUGGGAGUCACAGGG 17 downstream (SEQ ID NO: 926)CEP290-B1223 − ACGUUAUCAUUUUCCCA 17 upstream (SEQ ID NO: 927)CEP290-B1224 + ACUAAGACACUGCCAAU 17 downstream (SEQ ID NO: 603)CEP290-B1225 + AGAAAGAACACUGUGGU 17 downstream (SEQ ID NO: 928)CEP290-B1226 + AGACUGGAGAUAGAGAC 17 downstream (SEQ ID NO: 664)CEP290-B1227 + AGACUUAUAUUCCAUUA 17 downstream (SEQ ID NO: 651)CEP290-B1228 − AGAGACACAUUCAGUAA 17 upstream (SEQ ID NO: 754)CEP290-B1229 − AGAGUUCAAGCUAAUAC 17 downstream (SEQ ID NO: 931)CEP290-B1230 − AGAUCCUUUCACAGGAG 17 upstream (SEQ ID NO: 932)CEP290-B1231 + AGAUGAAAAAUACUCUU 17 upstream (SEQ ID NO: 718)CEP290-B1232 + AGAUGACAUGAGGUAAG 17 downstream (SEQ ID NO: 934)CEP290-B1233 − AGCAAGAGAUGAACUAG 17 downstream (SEQ ID NO: 935)CEP290-B1234 + AGCCUCUAUUUCUGAUG 17 upstream (SEQ ID NO: 709)CEP290-B1235 + AGGAAGAUGAACAAAUC 17 upstream (SEQ ID NO: 717)CEP290-B1236 + AGGACUUUCUAAUGCUG 17 upstream (SEQ ID NO: 938)CEP290-B1237 + AGGAGAUACUCAACACA 17 downstream (SEQ ID NO: 939)CEP290-B1238 + AGGAUAGGACAGAGGAC 17 upstream (SEQ ID NO: 940)CEP290-B1239 − AGGAUUCCUACUAAAUU 17 upstream (SEQ ID NO: 941)CEP290-B1240 − AGGAUUCCUACUAAAUU 17 upstream (SEQ ID NO: 941)CEP290-B1241 + AGGGUAGGAUUCAUGUU 17 downstream (SEQ ID NO: 942)CEP290-B1242 − AGUUCAAGCUAAUACAU 17 downstream (SEQ ID NO: 943)CEP290-B1243 + AUAAGCCUCUAUUUCUG 17 upstream (SEQ ID NO: 944)CEP290-B1244 − AUAAGUCUUUUGAUAUA 17 downstream (SEQ ID NO: 661)CEP290-B1245 + AUAAUUUAGUAGGAAUC 17 upstream (SEQ ID NO: 946)CEP290-B1246 + AUAAUUUAGUAGGAAUC 17 upstream (SEQ ID NO: 946)CEP290-B1247 − AUACCUUUUACUGAGGA 17 upstream (SEQ ID NO: 947)CEP290-B1248 − AUAGAGGCUUAUGGAUU 17 upstream (SEQ ID NO: 948)CEP290-B1249 + AUAGGACAGAGGACAUG 17 upstream (SEQ ID NO: 949)CEP290-B1250 − AUAUCUGUCUUCCUUAA 17 downstream (SEQ ID NO: 658)CEP290-B1251 − AUCAGAAAUAGAGGCUU 17 upstream (SEQ ID NO: 951)CEP290-B1252 + AUCAUUCUUGUGGCAGU 17 downstream (SEQ ID NO: 952)CEP290-B1253 − AUCCUCCUUACUGCCAC (SEQ 17 downstream ID NO: 953)CEP290-B1254 − AUCUAAGAUCCUUUCAC 17 upstream (SEQ ID NO: 696)CEP290-B1255 − AUCUUCCUCAUCAGAAA 17 upstream (SEQ ID NO: 955)CEP290-B1256 + AUGACAUGAGGUAAGUA 17 downstream (SEQ ID NO: 656)CEP290-B1257 + AUGACUCAUAAUUUAGU 17 upstream (SEQ ID NO: 704)CEP290-B1258 + AUGACUCAUAAUUUAGU 17 upstream (SEQ ID NO: 704)CEP290-B1259 − AUGAGAGUGAUUAGUGG 17 downstream (SEQ ID NO: 645)CEP290-B1260 − AUUAGAAAGUCCUAGGC 17 upstream (SEQ ID NO: 957)CEP290-B1261 + AUUCUUGUGGCAGUAAG 17 downstream (SEQ ID NO: 958)CEP290-B1262 + CACUCUCAUGUAUUAGC 17 downstream (SEQ ID NO: 959)CEP290-B1263 − CAUAUCUGUCUUCCUUA 17 downstream (SEQ ID NO: 960)CEP290-B1264 + CAUGACUCAUAAUUUAG 17 upstream (SEQ ID NO: 961)CEP290-B1265 + CAUGACUCAUAAUUUAG 17 upstream (SEQ ID NO: 961)CEP290-B1266 − CAUGAGAGUGAUUAGUG 17 downstream (SEQ ID NO: 962)CEP290-B1267 + CCUAGGACUUUCUAAUG 17 upstream (SEQ ID NO: 963)CEP290-B1268 − CCUAUCCUCUCCAGCAU (SEQ 17 upstream ID NO: 964)CEP290-B1269 + CUAGGACUUUCUAAUGC 17 upstream (SEQ ID NO: 695)CEP290-B1270 − CUCAUGUCAUCUAGAGC 17 downstream (SEQ ID NO: 966)CEP290-B1271 + CUCUUGCUCUAGAUGAC 17 downstream (SEQ ID NO: 967)CEP290-B1272 + CUCUUUGGCAAAAGCAG 17 upstream (SEQ ID NO: 968)CEP290-B1273 + CUCUUUGGCAAAAGCAG 17 upstream (SEQ ID NO: 968)CEP290-B1274 + CUGAACAAGUUUUGAAA 17 downstream (SEQ ID NO: 970)CEP290-B1275 + CUGAGCAAAACAACUGG 17 downstream (SEQ ID NO: 971)CEP290-B1276 − CUGCUGCUUUUGCCAAA 17 upstream (SEQ ID NO: 972)CEP290-B1277 − CUGCUGCUUUUGCCAAA 17 upstream (SEQ ID NO: 972)CEP290-B1278 + CUGGAGAGGAUAGGACA 17 upstream (SEQ ID NO: 973)CEP290-B1279 − UAAAUGAAAACGUUGUU 17 upstream (SEQ ID NO: 974)CEP290-B1280 − UAAAUGAAAACGUUGUU 17 upstream (SEQ ID NO: 974)CEP290-B1281 − UAAGCAUACUUUUUUUA 17 downstream (SEQ ID NO: 975)CEP290-B1282 + UAAGGAGGAUGUAAGAC 17 downstream (SEQ ID NO: 648)CEP290-B1283 + UAAUGCCUGAACAAGUU 17 downstream (SEQ ID NO: 976)CEP290-B1284 − UAAUUUGUUUCUGUGUG 17 downstream (SEQ ID NO: 977)CEP290-B1285 + UACAAAAGAACAUACAU 17 downstream (SEQ ID NO: 978)CEP290-B1286 − UAGGCAAGAGACAUCUU 17 upstream (SEQ ID NO: 979)CEP290-B1287 − UAUAAGUCUUUUGAUAU 17 downstream (SEQ ID NO: 980)CEP290-B1288 − UAUCUAAGAUCCUUUCA 17 upstream (SEQ ID NO: 981)CEP290-B1289 + UAUUUCUGAUGAGGAAG 17 upstream (SEQ ID NO: 982)CEP290-B1290 + UCACUGAGCAAAACAAC 17 downstream (SEQ ID NO: 650)CEP290-B1291 + UCAUUCUUGUGGCAGUA 17 downstream (SEQ ID NO: 2780)CEP290-B1292 − UCCAGUUGUUUUGCUCA 17 downstream (SEQ ID NO: 983)CEP290-B1293 + UCUAAUGCUGGAGAGGA 17 upstream (SEQ ID NO: 984)CEP290-B1294 + UGAACAAGUUUUGAAAC 17 downstream (SEQ ID NO: 659)CEP290-B1295 + UGAACUCUGUGCCAAAC 17 downstream (SEQ ID NO: 638)CEP290-B1296 + UGACAGUUUUUAAGGCG 17 downstream (SEQ ID NO: 642)CEP290-B1297 + UGAGCCAGCAAAAGCUU 17 upstream (SEQ ID NO: 752)CEP290-B1298 + UGCAGAACUAGUGUAGA 17 downstream (SEQ ID NO: 987)CEP290-B1299 + UGCCAAUAGGGAUAGGU 17 downstream (SEQ ID NO: 614)CEP290-B1300 − UGCUCUUUUCUAUAUAU 17 downstream (SEQ ID NO: 663)CEP290-B1301 + UGCUGGAGAGGAUAGGA 17 upstream (SEQ ID NO: 989)CEP290-B1302 − UGUCAAAUAUGGUGCUU 17 downstream (SEQ ID NO: 643)CEP290-B1303 − UGUUUAUCAAUAUUAUU 17 upstream (SEQ ID NO: 990)CEP290-B1304 − UGUUUAUCAAUAUUAUU 17 upstream (SEQ ID NO: 990)CEP290-B1305 + UUAAAAAAAGUAUGCUU 17 downstream (SEQ ID NO: 991)CEP290-B1306 + UUAAGGCGGGGAGUCAC 17 downstream (SEQ ID NO: 992)CEP290-B1307 + UUACUGAAUGUGUCUCU 17 upstream (SEQ ID NO: 993)CEP290-B1308 + UUACUGAAUGUGUCUCU 17 upstream (SEQ ID NO: 993)CEP290-B1309 + UUAUUCUACUCCUGUGA 17 upstream (SEQ ID NO: 768)CEP290-B1310 + UUCACUGAGCAAAACAA 17 downstream (SEQ ID NO: 995)CEP290-B1311 − UUCUGCUGCUUUUGCCA 17 upstream (SEQ ID NO: 996)CEP290-B1312 − UUCUGCUGCUUUUGCCA 17 upstream (SEQ ID NO: 996)CEP290-B1313 + UUGAACUCUGUGCCAAA 17 downstream (SEQ ID NO: 997)CEP290-B1314 + UUGACAGUUUUUAAGGC 17 downstream (SEQ ID NO: 654)CEP290-B1315 − UUGUGGAUAAUGUAUCA 17 upstream (SEQ ID NO: 999)CEP290-B1316 − UUGUUCAUCUUCCUCAU 17 upstream (SEQ ID NO: 1000)CEP290-B1317 − UUGUUCUGAGUAGCUUU 17 upstream (SEQ ID NO: 753)CEP290-B1318 + UUUGACAGUUUUUAAGG 17 downstream (SEQ ID NO: 662)CEP290-B1319 + UUUUGACAGUUUUUAAG 17 downstream (SEQ ID NO: 1003)

Table 6A provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the first tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, have good orthogonality, and start with G.It is contemplated herein that the targeting domain hybridizes to thetarget domain through complementary base pairing. Any of the targetingdomains in the table can be used with a N. meningitidis Cas9 moleculethat generates a double stranded break (Cas9 nuclease) or asingle-stranded break (Cas9 nickase).

TABLE 6A Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B65 − GAGUUCAAGCUAAUACAUGA 20 downstream(SEQ ID NO: 589) CEP290-B296 − GUUGUUCUGAGUAGCUU 17 upstream (SEQ ID NO:590) CEP290-B308 + GGCAAAAGCAGCAGAAAGCA 20 upstream (SEQ ID NO: 591)CEP290-B536 − GUUGUUCUGAGUAGCUU 17 upstream (SEQ ID NO: 590)CEP290-B482 + GGCAAAAGCAGCAGAAAGCA 20 upstream (SEQ ID NO: 591)

Table 6B provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the second tier parameters. Thetargeting domains are within 400 bp upstream of an Alu repeat or 700 bpdownstream of the mutation, have good orthogonality, and do not startwith G. It is contemplated herein that the targeting domain hybridizesto the target domain through complementary base pairing. Any of thetargeting domains in the table can be used with a N. meningitidis Cas9molecule that generates a double stranded break (Cas9 nuclease) or asingle-stranded break (Cas9 nickase).

TABLE 6B Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-B235 − UUCAAGCUAAUACAUGA 17 downstream(SEQ ID NO: 1004) CEP290-B109 + CACAUGGGAGUCACAGG 17 downstream (SEQ IDNO: 1005) CEP290-B129 + AGUCACAUGGGAGUCACAGG 20 downstream (SEQ ID NO:1006) CEP290-B295 − AAUAGAGGCUUAUGGAU 17 upstream (SEQ ID NO: 1007)CEP290-B297 − CUGAGGACAGAACAAGC 17 upstream (SEQ ID NO: 1008)CEP290-B298 − CAUCAGAAAUAGAGGCU 17 upstream (SEQ ID NO: 1009)CEP290-B299 − CUGCUUUUGCCAAAGAG 17 upstream (SEQ ID NO: 711)CEP290-B300 + AGCAGAAAGCAAACUGA 17 upstream (SEQ ID NO: 1011)CEP290-B301 + AAAAGCAGCAGAAAGCA 17 upstream (SEQ ID NO: 1012)CEP290-B302 − UUACUGAGGACAGAACAAGC 20 upstream (SEQ ID NO: 1013)CEP290-B303 − AACGUUGUUCUGAGUAGCUU 20 upstream (SEQ ID NO: 1014)CEP290-B304 − CUGCUGCUUUUGCCAAAGAG 20 upstream (SEQ ID NO: 725)CEP290-B305 − AGAAAUAGAGGCUUAUGGAU 20 upstream (SEQ ID NO: 1016)CEP290-B306 − CCUCAUCAGAAAUAGAGGCU 20 upstream (SEQ ID NO: 1017)CEP290-B307 + AGCAGCAGAAAGCAAACUGA 20 upstream (SEQ ID NO: 1018)CEP290-B531 − CUGCUUUUGCCAAAGAG 17 upstream (SEQ ID NO: 711)CEP290-B522 + AGCAGAAAGCAAACUGA 17 upstream (SEQ ID NO: 1011)CEP290-B537 + AAAAGCAGCAGAAAGCA 17 upstream (SEQ ID NO: 1012)CEP290-B504 − AACGUUGUUCUGAGUAGCUU 20 upstream (SEQ ID NO: 1014)CEP290-B478 − CUGCUGCUUUUGCCAAAGAG 20 upstream (SEQ ID NO: 725)CEP290-B526 + AGCAGCAGAAAGCAAACUGA 20 upstream (SEQ ID NO: 1018)

Table 7A provides targeting domains for introduction of an indel (e.g.,mediated by NHEJ) in close proximity to or including the LCA10 targetposition in the CEP290 gene selected according to the first tierparameters. The targeting domains are within 40 bases of the LCA10target position, have good orthogonality, start with G and PAM isNNGRRT. It is contemplated herein that in an embodiment the targetingdomain hybridizes to the target domain through complementary basepairing. Any of the targeting domains in the table can be used with a S.aureus Cas9 molecule that generates a double stranded break (Cas9nuclease) or a single-stranded break (Cas9 nickase).

TABLE 7A Target DNA Site gRNA Name Strand Targeting Domain LengthCEP290-12 − GCACCUGGCCCCAGUUGUAAUU 22 (SEQ ID NO: 398)

Table 7B provides targeting domains for introduction of an indel (e.g.,mediated by NHEJ) in close proximity to or including the LCA10 targetposition in the CEP290 gene selected according to the second tierparameters. The targeting domains are within 40 bases of the LCA10target position, have good orthogonality, and PAM is NNGRRT. It iscontemplated herein that the targeting domain hybridizes to the targetdomain through complementary base pairing. Any of the targeting domainsin the table can be used with a S. aureus Cas9 molecule that generates adouble stranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 7B Target DNA Site gRNA Name Strand Targeting Domain LengthCEP290-35 + AAAUAAAACUAAGACACUGCCAAU 24 (SEQ ID NO: 1025) CEP290-36 +AAUAAAACUAAGACACUGCCAAU 23 (SEQ ID NO: 1026) CEP290-37 +AUAAAACUAAGACACUGCCAAU 22 (SEQ ID NO: 1027) CEP290-38 +AAAACUAAGACACUGCCAAU (SEQ 20 ID NO: 610) CEP290-39 + AAACUAAGACACUGCCAAU(SEQ 19 ID NO: 1028) CEP290-40 + AACUAAGACACUGCCAAU (SEQ ID 18 NO: 1029)CEP290-512 − ACCUGGCCCCAGUUGUAAUU (SEQ 20 ID NO: 616) CEP290-17 −CCGCACCUGGCCCCAGUUGUAAUU 24 (SEQ ID NO: 1030) CEP290-41 −CGCACCUGGCCCCAGUUGUAAUU 23 (SEQ ID NO: 1031) CEP290-42 −CACCUGGCCCCAGUUGUAAUU 21 (SEQ ID NO: 1032) CEP290-513 −CCUGGCCCCAGUUGUAAUU (SEQ 19 ID NO: 1033) CEP290-514 − CUGGCCCCAGUUGUAAUU(SEQ ID 18 NO: 1034) CEP290-43 + UAAAACUAAGACACUGCCAAU 21 (SEQ ID NO:1035)

Table 7C provides targeting domains for introduction of an indel (e.g.,mediated by NHEJ) in close proximity to or including the LCA10 targetposition in the CEP290 gene selected according to the fifth tierparameters. The targeting domains are within 40 bases of the LCA10target position, and PAM is NNGRRV. It is contemplated herein that thetargeting domain hybridizes to the target domain through complementarybase pairing. Any of the targeting domains in the table can be used witha S. aureus Cas9 molecule that generates a double stranded break (Cas9nuclease) or a single-stranded break (Cas9 nickase).

TABLE 7C Target DNA Site gRNA Name Strand Targeting Domain LengthCEP290-44 + AAAAUAAAACUAAGACACUGCCAA 24 (SEQ ID NO: 1036) CEP290-45 +AAAUAAAACUAAGACACUGCCAA 23 (SEQ ID NO: 1037) CEP290-46 +AAUAAAACUAAGACACUGCCAA 22 (SEQ ID NO: 1038) CEP290-47 +AUAAAACUAAGACACUGCCAA 21 (SEQ ID NO: 1039) CEP290-48 +AAAACUAAGACACUGCCAA (SEQ 19 ID NO: 1040) CEP290-49 + AAACUAAGACACUGCCAA(SEQ ID 18 NO: 1041) CEP290-16 + AAGACACUGCCAAUAGGGAUAGGU 24 (SEQ ID NO:1042) CEP290-50 + AGACACUGCCAAUAGGGAUAGGU 23 (SEQ ID NO: 1043)CEP290-51 + ACACUGCCAAUAGGGAUAGGU 21 (SEQ ID NO: 1044) CEP290-510 +ACUGCCAAUAGGGAUAGGU (SEQ 19 ID NO: 1045) CEP290-509 +CACUGCCAAUAGGGAUAGGU (SEQ 20 ID NO: 613) CEP290-511 + CUGCCAAUAGGGAUAGGU(SEQ ID 18 NO: 1046) CEP290-11 + GACACUGCCAAUAGGGAUAGGU 22 (SEQ ID NO:1047) CEP290-52 + UAAAACUAAGACACUGCCAA (SEQ 20 ID NO: 871) CEP290-13 +AUGAGAUACUCACAAUUACAAC 22 (SEQ ID NO: 1049) CEP290-53 +AGAUACUCACAAUUACAAC (SEQ 19 ID NO: 1050) CEP290-18 +GUAUGAGAUACUCACAAUUACAAC 24 (SEQ ID NO: 1051) CEP290-54 +GAGAUACUCACAAUUACAAC (SEQ 20 ID NO: 395) CEP290-55 + GAUACUCACAAUUACAAC(SEQ ID 18 NO: 1052) CEP290-14 + UAUGAGAUACUCACAAUUACAAC 23 (SEQ ID NO:1053) CEP290-57 + UGAGAUACUCACAAUUACAAC 21 (SEQ ID NO: 1054) CEP290-58 +AUGAGAUAUUCACAAUUACAA 21 (SEQ ID NO: 1055) CEP290-59 +AGAUAUUCACAAUUACAA (SEQ ID 18 NO: 1056) CEP290-19 +GGUAUGAGAUAUUCACAAUUACAA 24 (SEQ ID NO: 1057) CEP290-61 +GUAUGAGAUAUUCACAAUUACAA 23 (SEQ ID NO: 1058) CEP290-63 +GAGAUAUUCACAAUUACAA (SEQ 19 ID NO: 1059) CEP290-65 +UAUGAGAUAUUCACAAUUACAA 22 (SEQ ID NO: 1060) CEP290-66 +UGAGAUAUUCACAAUUACAA (SEQ 20 ID NO: 1061)

Table 7D provides targeting domains for introduction of an indel (e.g.,mediated by NHEJ) in close proximity to or including the LCA10 targetposition in the CEP290 gene that can be used for dual targeting. Any ofthe targeting domains in the table can be used with a S. aureus Cas9(nickase) molecule to generate a single stranded break. Exemplarynickase pairs including selecting a targeting domain from Group A and asecond targeting domain from Group B. It is contemplated herein that atargeting domain of Group A can be combined with any of the targetingdomains of Group B. For example, the CEP290-12 or CEP290-17 can becombined with CEP290-11 or CEP290-16.

TABLE 7D Group A Group B CEP290-12 CEP290-11 CEP290-17 CEP290-16

Table 8A provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the first tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation, havegood orthogonality, and start with G. It is contemplated herein that thetargeting domain hybridizes to the target domain through complementarybase pairing. Any of the targeting domains in the table can be used witha S. pyogenes Cas9 molecule that generates a double stranded break (Cas9nuclease) or a single-stranded break (Cas9 nickase).

TABLE 8A Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-67 + GAAAGAUGAAAAAUACUCUU 20 upstream (SEQID NO: 462) CEP290-68 − GAAAUAGAUGUAGAUUG 17 downstream (SEQ ID NO: 463)CEP290-70 − GAAAUAUUAAGGGCUCUUCC 20 upstream (SEQ ID NO: 464)CEP290-71 + GAACAAAAGCCAGGGACCAU 20 upstream (SEQ ID NO: 465) CEP290-72− GAACUCUAUACCUUUUACUG 20 upstream (SEQ ID NO: 466) CEP290-73 −GAAGAAUGGAAUAGAUAAUA 20 downstream (SEQ ID NO: 467) CEP290-74 +GAAUAGUUUGUUCUGGGUAC 20 upstream (SEQ ID NO: 468) CEP290-75 −GAAUGGAAUAGAUAAUA 17 downstream (SEQ ID NO: 469) CEP290-76 +GAAUUUACAGAGUGCAUCCA 20 upstream (SEQ ID NO: 470) CEP290-77 −GAGAAAAAGGAGCAUGAAAC 20 upstream (SEQ ID NO: 471) CEP290-78 −GAGAGCCACAGUGCAUG 17 downstream (SEQ ID NO: 472) CEP290-79 −GAGGUAGAAUCAAGAAG 17 downstream (SEQ ID NO: 473) CEP290-80 +GAGUGCAUCCAUGGUCC 17 upstream (SEQ ID NO: 474) CEP290-81 +GAUAACUACAAAGGGUC 17 upstream (SEQ ID NO: 475) CEP290-82 +GAUAGAGACAGGAAUAA 17 downstream (SEQ ID NO: 476) CEP290-83 +GAUGAAAAAUACUCUUU 17 upstream (SEQ ID NO: 477) CEP290-84 +GAUGACAUGAGGUAAGU 17 downstream (SEQ ID NO: 478) CEP290-85 +GAUGCAGAACUAGUGUAGAC 20 downstream (SEQ ID NO: 460) CEP290-86 +GCAGAACUAGUGUAGAC 17 downstream (SEQ ID NO: 458) CEP290-87 −GCAUGUGGUGUCAAAUA 17 downstream (SEQ ID NO: 479) CEP290-88 +GCCUGAACAAGUUUUGAAAC 20 downstream (SEQ ID NO: 480) CEP290-89 −GCUACCGGUUACCUGAA 17 downstream (SEQ ID NO: 457) CEP290-90 −GCUCUUUUCUAUAUAUA 17 downstream (SEQ ID NO: 481) CEP290-91 +GCUUGAACUCUGUGCCAAAC 20 downstream (SEQ ID NO: 461) CEP290-92 +GCUUUUGACAGUUUUUAAGG 20 downstream (SEQ ID NO: 482) CEP290-93 −GCUUUUGUUCCUUGGAA 17 upstream (SEQ ID NO: 483) CEP290-94 +GGAACAAAAGCCAGGGACCA 20 upstream (SEQ ID NO: 484) CEP290-95 +GGACUUGACUUUUACCCUUC 20 downstream (SEQ ID NO: 485) CEP290-96 +GGAGAAUAGUUUGUUCU 17 upstream (SEQ ID NO: 486) CEP290-97 +GGAGUCACAUGGGAGUCACA 20 downstream (SEQ ID NO: 487) CEP290-98 +GGAUAGGACAGAGGACA 17 upstream (SEQ ID NO: 488) CEP290-99 +GGCUGUAAGAUAACUACAAA 20 upstream (SEQ ID NO: 489) CEP290-100 +GGGAGAAUAGUUUGUUC 17 upstream (SEQ ID NO: 490) CEP290-101 +GGGAGUCACAUGGGAGUCAC 20 downstream (SEQ ID NO: 491) CEP290-102 −GGGCUCUUCCUGGACCA (SEQ 17 upstream ID NO: 492) CEP290-103 +GGGUACAGGGGUAAGAGAAA 20 upstream (SEQ ID NO: 493) CEP290-104 −GGUCCCUGGCUUUUGUUCCU 20 upstream (SEQ ID NO: 494) CEP290-105 −GUAAAGGUUCAUGAGACUAG 20 downstream (SEQ ID NO: 495) CEP290-106 +GUAACAUAAUCACCUCUCUU 20 upstream (SEQ ID NO: 496) CEP290-107 +GUAAGACUGGAGAUAGAGAC 20 downstream (SEQ ID NO: 497) CEP290-108 +GUACAGGGGUAAGAGAA 17 upstream (SEQ ID NO: 498) CEP290-109 +GUAGCUUUUGACAGUUUUUA 20 downstream (SEQ ID NO: 499) CEP290-110 +GUCACAUGGGAGUCACA 17 downstream (SEQ ID NO: 500) CEP290-111 −GUGGAGAGCCACAGUGCAUG 20 downstream (SEQ ID NO: 501) CEP290-112 −GUUACAAUCUGUGAAUA 17 upstream (SEQ ID NO: 502) CEP290-113 +GUUCUGUCCUCAGUAAA 17 upstream (SEQ ID NO: 503) CEP290-114 −GUUGAGUAUCUCCUGUU 17 downstream (SEQ ID NO: 459) CEP290-115 +GUUUAGAAUGAUCAUUCUUG 20 downstream (SEQ ID NO: 504) CEP290-116 +GUUUGUUCUGGGUACAG 17 upstream (SEQ ID NO: 505) CEP290-117 −UAAAAACUGUCAAAAGCUAC 20 downstream (SEQ ID NO: 506) CEP290-118 +UAAAAGGUAUAGAGUUCAAG 20 upstream (SEQ ID NO: 507) CEP290-119 +UAAAUCAUGCAAGUGACCUA 20 upstream (SEQ ID NO: 508) CEP290-120 +UAAGAUAACUACAAAGGGUC 20 upstream (SEQ ID NO: 509)

Table 8B provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the second tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation, havegood orthogonality, and do not start with G. It is contemplated hereinthat the targeting domain hybridizes to the target domain throughcomplementary base pairing. Any of the targeting domains in the tablecan be used with a S. pyogenes Cas9 molecule that generates a doublestranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 8B Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-121 − AAAAAGGAGCAUGAAAC 17 upstream (SEQID NO: 1062) CEP290-122 + AAAACUAAGACACUGCCAAU 20 downstream (SEQ ID NO:610) CEP290-123 + AAAAGACUUAUAUUCCAUUA 20 downstream (SEQ ID NO: 685)CEP290-124 − AAAAGCUACCGGUUACCUGA 20 downstream (SEQ ID NO: 621)CEP290-125 − AAAAUUAUGCCUAUUUAGUG 20 upstream (SEQ ID NO: 729)CEP290-126 + AAACAACUGGAAGAGAGAAA 20 downstream (SEQ ID NO: 691)CEP290-127 + AAACUAAGACACUGCCAAUA 20 downstream (SEQ ID NO: 609)CEP290-128 − AAACUGUCAAAAGCUAC 17 downstream (SEQ ID NO: 655) CEP290-129− AAAGAAAUAGAUGUAGAUUG 20 downstream (SEQ ID NO: 1066) CEP290-130 +AAAGAUGAAAAAUACUCUUU 20 upstream (SEQ ID NO: 737) CEP290-131 −AAAGCUACCGGUUACCUGAA 20 downstream (SEQ ID NO: 620) CEP290-133 −AAAUAGAGGCUUAUGGAUUU 20 upstream (SEQ ID NO: 731) CEP290-134 +AAAUUAUCACCACACUAAAU 20 upstream (SEQ ID NO: 727) CEP290-135 −AACAAACUAUUCUCCCA (SEQ 17 upstream ID NO: 1070) CEP290-136 −AACAGUAGCUGAAAUAUUAA 20 upstream (SEQ ID NO: 1071) CEP290-137 +AACAUGACUCAUAAUUUAGU 20 upstream (SEQ ID NO: 721) CEP290-138 −AACUAUUCUCCCAUGGUCCC 20 upstream (SEQ ID NO: 1073) CEP290-140 +AAGACACUGCCAAUAGGGAU 20 downstream (SEQ ID NO: 600) CEP290-141 −AAGGAAAUACAAAAACUGGA 20 downstream (SEQ ID NO: 1074) CEP290-142 +AAGGUAUAGAGUUCAAG 17 upstream (SEQ ID NO: 1075) CEP290-143 −AAGGUUCAUGAGACUAG 17 downstream (SEQ ID NO: 1076) CEP290-144 +AAUAGUUUGUUCUGGGUACA 20 upstream (SEQ ID NO: 1077) CEP290-145 −AAUAUAAGUCUUUUGAUAUA 20 downstream (SEQ ID NO: 687) CEP290-146 −AAUAUAUUAUCUAUUUAUAG 20 upstream (SEQ ID NO: 1079) CEP290-147 −AAUAUUGUAAUCAAAGG 17 upstream (SEQ ID NO: 1080) CEP290-148 +AAUAUUUCAGCUACUGU 17 upstream (SEQ ID NO: 1081) CEP290-149 −AAUUAUUGUUGCUUUUUGAG 20 downstream (SEQ ID NO: 1082) CEP290-150 +AAUUCACUGAGCAAAACAAC 20 downstream (SEQ ID NO: 678) CEP290-151 +ACAAAAGCCAGGGACCA 17 upstream (SEQ ID NO: 1084) CEP290-152 +ACACUGCCAAUAGGGAU 17 downstream (SEQ ID NO: 595) CEP290-153 +ACAGAGUGCAUCCAUGGUCC 20 upstream (SEQ ID NO: 1085) CEP290-154 +ACAUAAUCACCUCUCUU (SEQ 17 upstream ID NO: 712) CEP290-155 −ACCAGACAUCUAAGAGAAAA 20 upstream (SEQ ID NO: 1087) CEP290-156 −ACGUGCUCUUUUCUAUAUAU 20 downstream (SEQ ID NO: 622) CEP290-157 +ACUUUCUAAUGCUGGAG 17 upstream (SEQ ID NO: 700) CEP290-158 +ACUUUUACCCUUCAGGUAAC 20 downstream (SEQ ID NO: 626) CEP290-159 −AGAAUAUUGUAAUCAAAGGA 20 upstream (SEQ ID NO: 1089) CEP290-160 −AGACAUCUAAGAGAAAA 17 upstream (SEQ ID NO: 1090) CEP290-161 +AGACUUAUAUUCCAUUA 17 downstream (SEQ ID NO: 651) CEP290-162 +AGAGGAUAGGACAGAGGACA 20 upstream (SEQ ID NO: 735) CEP290-163 +AGAUGACAUGAGGUAAGUAG 20 downstream (SEQ ID NO: 677) CEP290-164 +AGAUGUCUGGUUAAAAG 17 upstream (SEQ ID NO: 1093) CEP290-165 +AGCCUCUAUUUCUGAUG 17 upstream (SEQ ID NO: 709) CEP290-166 −AGCUACCGGUUACCUGA 17 downstream (SEQ ID NO: 618) CEP290-167 −AGCUCAAAAGCUUUUGC 17 upstream (SEQ ID NO: 698) CEP290-168 −AGGAAAUACAAAAACUGGAU 20 downstream (SEQ ID NO: 1096) CEP290-169 +AGGAAGAUGAACAAAUC 17 upstream (SEQ ID NO: 717) CEP290-170 +AGGACAGAGGACAUGGAGAA 20 upstream (SEQ ID NO: 736) CEP290-171 +AGGACUUUCUAAUGCUGGAG 20 upstream (SEQ ID NO: 719) CEP290-172 −AGGCAAGAGACAUCUUG 17 upstream (SEQ ID NO: 708) CEP290-173 −AGGUAGAAUAUUGUAAUCAA 20 upstream (SEQ ID NO: 1101) CEP290-174 −AGUAGCUGAAAUAUUAA 17 upstream (SEQ ID NO: 1102) CEP290-175 +AGUCACAUGGGAGUCAC 17 downstream (SEQ ID NO: 644) CEP290-176 −AGUGCAUGUGGUGUCAAAUA 20 downstream (SEQ ID NO: 627) CEP290-177 +AGUUUGUUCUGGGUACA 17 upstream (SEQ ID NO: 1103) CEP290-178 +AUAAGCCUCUAUUUCUGAUG 20 upstream (SEQ ID NO: 723) CEP290-179 −AUAAGUCUUUUGAUAUA 17 downstream (SEQ ID NO: 661) CEP290-180 +AUACAUAAGAAAGAACACUG 20 downstream (SEQ ID NO: 686) CEP290-181 +AUAGUUUGUUCUGGGUACAG 20 upstream (SEQ ID NO: 1107) CEP290-182 −AUAUCUGUCUUCCUUAA 17 downstream (SEQ ID NO: 658) CEP290-183 −AUAUUAAGGGCUCUUCC 17 upstream (SEQ ID NO: 1109) CEP290-184 −AUAUUGUAAUCAAAGGA 17 upstream (SEQ ID NO: 1110) CEP290-185 +AUCAUGCAAGUGACCUA 17 upstream (SEQ ID NO: 1111) CEP290-186 −AUCUAAGAUCCUUUCAC 17 upstream (SEQ ID NO: 696) CEP290-187 −AUCUUCCUCAUCAGAAAUAG 20 upstream (SEQ ID NO: 722) CEP290-188 +AUGACAUGAGGUAAGUA 17 downstream (SEQ ID NO: 656) CEP290-189 +AUGACUCAUAAUUUAGU 17 upstream (SEQ ID NO: 704) CEP290-190 −AUGAGAGUGAUUAGUGG 17 downstream (SEQ ID NO: 645) CEP290-191 +AUGAGGAAGAUGAACAAAUC 20 upstream (SEQ ID NO: 733) CEP290-192 +AUGGGAGAAUAGUUUGUUCU 20 upstream (SEQ ID NO: 1116) CEP290-193 −AUUAGCUCAAAAGCUUUUGC 20 upstream (SEQ ID NO: 633) CEP290-194 −AUUAUGCCUAUUUAGUG 17 upstream (SEQ ID NO: 703) CEP290-195 +AUUCCAAGGAACAAAAGCCA 20 upstream (SEQ ID NO: 1118) CEP290-196 −AUUGAGGUAGAAUCAAGAAG 20 downstream (SEQ ID NO: 1119) CEP290-197 +AUUUGACACCACAUGCACUG 20 downstream (SEQ ID NO: 623) CEP290-198 +CAAAAGCCAGGGACCAU 17 upstream (SEQ ID NO: 1120) CEP290-199 −CAACAGUAGCUGAAAUAUUA 20 upstream (SEQ ID NO: 1121) CEP290-200 +CAAGAUGUCUCUUGCCU 17 upstream (SEQ ID NO: 702) CEP290-201 −CAGAACAAACUAUUCUCCCA 20 upstream (SEQ ID NO: 1123) CEP290-202 −CAGAUUUCAUGUGUGAAGAA 20 downstream (SEQ ID NO: 1124) CEP290-204 −CAGCAUUAGAAAGUCCU 17 upstream (SEQ ID NO: 710) CEP290-205 +CAGGGGUAAGAGAAAGGGAU 20 upstream (SEQ ID NO: 1126) CEP290-206 +CAGUAAGGAGGAUGUAAGAC 20 downstream (SEQ ID NO: 676) CEP290-207 −CAGUAGCUGAAAUAUUA 17 upstream (SEQ ID NO: 1128) CEP290-208 +CAUAAGAAAGAACACUG 17 downstream (SEQ ID NO: 665) CEP290-209 +CAUGGGAGAAUAGUUUGUUC 20 upstream (SEQ ID NO: 1130) CEP290-210 +CAUGGGAGUCACAGGGU 17 downstream (SEQ ID NO: 652) CEP290-211 +CAUUCCAAGGAACAAAAGCC 20 upstream (SEQ ID NO: 1131) CEP290-212 +CCACAAGAUGUCUCUUGCCU 20 upstream (SEQ ID NO: 630) CEP290-213 −CCUAGGCAAGAGACAUCUUG 20 upstream (SEQ ID NO: 631) CEP290-214 −CGUGCUCUUUUCUAUAUAUA 20 downstream (SEQ ID NO: 624) CEP290-215 −CGUUGUUCUGAGUAGCUUUC 20 upstream (SEQ ID NO: 629) CEP290-216 +CUAAGACACUGCCAAUA 17 downstream (SEQ ID NO: 597) CEP290-217 +CUAAUGCUGGAGAGGAU 17 upstream (SEQ ID NO: 707) CEP290-218 +CUAGAUGACAUGAGGUAAGU 20 downstream (SEQ ID NO: 671) CEP290-219 +CUAGGACUUUCUAAUGC 17 upstream (SEQ ID NO: 695) CEP290-220 −CUCAUACCUAUCCCUAU (SEQ 17 downstream ID NO: 594) CEP290-221 −CUCCAGCAUUAGAAAGUCCU 20 upstream (SEQ ID NO: 720) CEP290-222 −CUCUAUACCUUUUACUG 17 upstream (SEQ ID NO: 701) CEP290-223 +CUCUUGCUCUAGAUGACAUG 20 downstream (SEQ ID NO: 675) CEP290-224 −CUGCUGCUUUUGCCAAAGAG 20 upstream (SEQ ID NO: 725) CEP290-225 −CUGCUUUUGCCAAAGAG 17 upstream (SEQ ID NO: 711) CEP290-226 −CUGGCUUUUGUUCCUUGGAA 20 upstream (SEQ ID NO: 1140) CEP290-227 +CUGUAAGAUAACUACAA 17 upstream (SEQ ID NO: 1141) CEP290-228 −CUUAAGCAUACUUUUUUUAA 20 downstream (SEQ ID NO: 690) CEP290-229 +CUUAAUAUUUCAGCUACUGU 20 upstream (SEQ ID NO: 1143) CEP290-231 +CUUAGAUGUCUGGUUAAAAG 20 upstream (SEQ ID NO: 1144) CEP290-232 −CUUAUCUAAGAUCCUUUCAC 20 upstream (SEQ ID NO: 734) CEP290-233 +CUUGACUUUUACCCUUC (SEQ 17 downstream ID NO: 649) CEP290-234 +CUUGUUCUGUCCUCAGUAAA 20 upstream (SEQ ID NO: 728) CEP290-235 +CUUUUGACAGUUUUUAAGGC 20 downstream (SEQ ID NO: 684)

Table 8C provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the third tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation, andstart with G. It is contemplated herein that the targeting domainhybridizes to the target domain through complementary base pairing. Anyof the targeting domains in the table can be used with a S. pyogenesCas9 molecule that generates a double stranded break (Cas9 nuclease) ora single-stranded break (Cas9 nickase).

TABLE 8C Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-236 − GAAAUACAAAAACUGGA 17 downstream (SEQID NO: 1148) CEP290-237 + GCUUUUGACAGUUUUUA 17 downstream (SEQ ID NO:634) CEP290-238 + GGAGAUAGAGACAGGAAUAA 20 downstream (SEQ ID NO: 635)CEP290-239 − GGAGUGCAGUGGAGUGAUCU 20 downstream (SEQ ID NO: 1149)CEP290-240 + GGGGUAAGAGAAAGGGA 17 upstream (SEQ ID NO: 1150)CEP290-241 + GGGUAAGAGAAAGGGAU 17 upstream (SEQ ID NO: 1151) CEP290-242− GUCUCACUGUGUUGCCC (SEQ 17 downstream ID NO: 1152) CEP290-243 −GUGCAGUGGAGUGAUCU 17 downstream (SEQ ID NO: 1153) CEP290-244 +GUGUGUGUGUGUGUGUUAUG 20 upstream (SEQ ID NO: 1154) CEP290-245 +GUGUGUGUGUGUUAUGU 17 upstream (SEQ ID NO: 1155)

Table 8D provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the fourth tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation, anddo not start with G. It is contemplated herein that the targeting domainhybridizes to the target domain through complementary base pairing. Anyof the targeting domains in the table can be used with a S. pyogenesCas9 molecule that generates a double stranded break (Cas9 nuclease) ora single-stranded break (Cas9 nickase).

TABLE 8D Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-246 − AAAUACAAAAACUGGAU 17 downstream (SEQID NO: 1156) CEP290-247 − AAGCAUACUUUUUUUAA 17 downstream (SEQ ID NO:667) CEP290-248 + AAGGCGGGGAGUCACAU 17 downstream (SEQ ID NO: 636)CEP290-249 + AAGUAUGCUUAAGAAAAAAA 20 downstream (SEQ ID NO: 693)CEP290-250 + ACAGAGGACAUGGAGAA 17 upstream (SEQ ID NO: 715) CEP290-251 +ACAGGGGUAAGAGAAAGGGA 20 upstream (SEQ ID NO: 1160) CEP290-253 +ACUAAGACACUGCCAAU 17 downstream (SEQ ID NO: 603) CEP290-254 +ACUCCACUGCACUCCAGCCU 20 downstream (SEQ ID NO: 1161) CEP290-255 +AGACUGGAGAUAGAGAC 17 downstream (SEQ ID NO: 664) CEP290-256 −AGAGUCUCACUGUGUUGCCC 20 downstream (SEQ ID NO: 1163) CEP290-257 +AGAUGAAAAAUACUCUU 17 upstream (SEQ ID NO: 718) CEP290-258 −AUAUUAUCUAUUUAUAG 17 upstream (SEQ ID NO: 1165) CEP290-259 −AUUUCAUGUGUGAAGAA 17 downstream (SEQ ID NO: 1166) CEP290-260 −AUUUUUUAUUAUCUUUAUUG 20 downstream (SEQ ID NO: 694) CEP290-261 +CAACUGGAAGAGAGAAA 17 downstream (SEQ ID NO: 668) CEP290-262 +CACUCCACUGCACUCCAGCC 20 downstream (SEQ ID NO: 1169) CEP290-263 −CACUGUGUUGCCCAGGC (SEQ 17 downstream ID NO: 1170) CEP290-264 +CCAAGGAACAAAAGCCA 17 upstream (SEQ ID NO: 1171) CEP290-265 +CCACUGCACUCCAGCCU (SEQ 17 downstream ID NO: 1172) CEP290-266 −CCCAGGCUGGAGUGCAG 17 downstream (SEQ ID NO: 1173) CEP290-267 −CCCUGGCUUUUGUUCCU (SEQ 17 upstream ID NO: 1174) CEP290-268 +CGCUUGAACCUGGGAGGCAG 20 downstream (SEQ ID NO: 1175) CEP290-269 −UAAGGAAAUACAAAAAC 17 downstream (SEQ ID NO: 1176) CEP290-270 −UAAUAAGGAAAUACAAAAAC 20 downstream (SEQ ID NO: 1177) CEP290-271 −UACUGCAACCUCUGCCUCCC 20 downstream (SEQ ID NO: 1178) CEP290-272 +UAUGCUUAAGAAAAAAA 17 downstream (SEQ ID NO: 669) CEP290-273 +UCAUUCUUGUGGCAGUAAGG 20 downstream (SEQ ID NO: 692) CEP290-274 +UCCACUGCACUCCAGCC (SEQ 17 downstream ID NO: 1181) CEP290-275 −UCUCACUGUGUUGCCCAGGC 20 downstream (SEQ ID NO: 1182) CEP290-276 +UGAACAAGUUUUGAAAC 17 downstream (SEQ ID NO: 659) CEP290-277 −UGCAACCUCUGCCUCCC (SEQ 17 downstream ID NO: 1184) CEP290-278 +UGUGUGUGUGUGUGUUAUGU 20 upstream (SEQ ID NO: 1185) CEP290-279 +UGUGUGUGUGUGUUAUG 17 upstream (SEQ ID NO: 1186) CEP290-280 +UUCUUGUGGCAGUAAGG 17 downstream (SEQ ID NO: 666) CEP290-281 +UUGAACCUGGGAGGCAG 17 downstream (SEQ ID NO: 1188) CEP290-282 −UUGCCCAGGCUGGAGUGCAG 20 downstream (SEQ ID NO: 1189) CEP290-283 −UUUUAUUAUCUUUAUUG 17 downstream (SEQ ID NO: 670)

Table 9A provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the first tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation, havegood orthogonality, start with G and PAM is NNGRRT. It is contemplatedherein that the targeting domain hybridizes to the target domain throughcomplementary base pairing. Any of the targeting domains in the tablecan be used with a S. aureus Cas9 molecule that generates a doublestranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 9A 1st Tier Target Position DNA Site relative to gRNA Name StrandTargeting Domain Length mutation CEP290-284 + GCUAAAUCAUGCAAGUGACCUAAG24 upstream (SEQ ID NO: 511) CEP290-487 − GGUCACUUGCAUGAUUUAG (SEQ ID 19upstream NO: 512) CEP290-486 − GUCACUUGCAUGAUUUAG (SEQ ID 18 upstreamNO: 513) CEP290-285 + GCCUAGGACUUUCUAAUGCUGGA 23 upstream (SEQ ID NO:514) CEP290-479 + GGACUUUCUAAUGCUGGA (SEQ ID 18 upstream NO: 515)CEP290-286 + GGGACCAUGGGAGAAUAGUUUGUU 24 upstream (SEQ ID NO: 516)CEP290-287 + GGACCAUGGGAGAAUAGUUUGUU 23 upstream (SEQ ID NO: 517)CEP290-288 + GACCAUGGGAGAAUAGUUUGUU 22 upstream (SEQ ID NO: 518)CEP290-289 − GGUCCCUGGCUUUUGUUCCUUGGA 24 upstream (SEQ ID NO: 519)CEP290-290 − GUCCCUGGCUUUUGUUCCUUGGA 23 upstream (SEQ ID NO: 520)CEP290-374 − GAAAACGUUGUUCUGAGUAGCUUU 24 upstream (SEQ ID NO: 521)CEP290-478 − GUUGUUCUGAGUAGCUUU (SEQ ID 18 upstream NO: 522) CEP290-489− GGUCCCUGGCUUUUGUUCCU (SEQ 20 upstream ID NO: 494) CEP290-488 −GUCCCUGGCUUUUGUUCCU (SEQ ID 19 upstream NO: 523) CEP290-291 −GACAUCUUGUGGAUAAUGUAUCA 23 upstream (SEQ ID NO: 524) CEP290-292 −GUCCUAGGCAAGAGACAUCUU 21 upstream (SEQ ID NO: 525) CEP290-293 +GCCAGCAAAAGCUUUUGAGCUAA 23 upstream (SEQ ID NO: 526) CEP290-481 +GCAAAAGCUUUUGAGCUAA (SEQ ID 19 upstream NO: 527) CEP290-294 +GAUCUUAUUCUACUCCUGUGA 21 upstream (SEQ ID NO: 528) CEP290-295 −GCUUUCAGGAUUCCUACUAAAUU 23 upstream (SEQ ID NO: 529) CEP290-323 +GUUCUGUCCUCAGUAAAAGGUA 22 upstream (SEQ ID NO: 530) CEP290-480 +GAACAACGUUUUCAUUUA (SEQ ID 18 upstream NO: 531) CEP290-296 −GUAGAAUAUCAUAAGUUACAAUCU 24 upstream (SEQ ID NO: 532) CEP290-297 −GAAUAUCAUAAGUUACAAUCU 21 upstream (SEQ ID NO: 533) CEP290-298 +GUGGCUGUAAGAUAACUACA (SEQ 20 upstream ID NO: 534) CEP290-299 +GGCUGUAAGAUAACUACA (SEQ ID 18 upstream NO: 535) CEP290-300 −GUUUAACGUUAUCAUUUUCCCA 22 upstream (SEQ ID NO: 536) CEP290-301 +GUAAGAGAAAGGGAUGGGCACUUA 24 upstream (SEQ ID NO: 537) CEP290-492 +GAGAAAGGGAUGGGCACUUA (SEQ 20 upstream ID NO: 538) CEP290-491 +GAAAGGGAUGGGCACUUA (SEQ ID 18 upstream NO: 539) CEP290-483 −GUAAAUGAAAACGUUGUU (SEQ ID 18 upstream NO: 540) CEP290-302 +GAUAAACAUGACUCAUAAUUUAGU 24 upstream (SEQ ID NO: 541) CEP290-303 +GGAACAAAAGCCAGGGACCAUGG 23 upstream (SEQ ID NO: 542) CEP290-304 +GAACAAAAGCCAGGGACCAUGG 22 upstream (SEQ ID NO: 543) CEP290-305 +GGGAGAAUAGUUUGUUCUGGGUAC 24 upstream (SEQ ID NO: 544) CEP290-306 +GGAGAAUAGUUUGUUCUGGGUAC 23 upstream (SEQ ID NO: 545) CEP290-307 +GAGAAUAGUUUGUUCUGGGUAC 22 upstream (SEQ ID NO: 546) CEP290-490 +GAAUAGUUUGUUCUGGGUAC (SEQ 20 upstream ID NO: 468) CEP290-482 −GAAAUAGAGGCUUAUGGAUU (SEQ 20 upstream ID NO: 547) CEP290-308 +GUUCUGGGUACAGGGGUAAGAGAA 24 upstream (SEQ ID NO: 548) CEP290-494 +GGGUACAGGGGUAAGAGAA (SEQ 19 upstream ID NO: 549) CEP290-493 +GGUACAGGGGUAAGAGAA (SEQ ID 18 upstream NO: 550) CEP290-309 −GUAAAUUCUCAUCAUUUUUUAUUG 24 upstream (SEQ ID NO: 551) CEP290-310 +GGAGAGGAUAGGACAGAGGACAUG 24 upstream (SEQ ID NO: 552) CEP290-311 +GAGAGGAUAGGACAGAGGACAUG 23 upstream (SEQ ID NO: 553) CEP290-313 +GAGGAUAGGACAGAGGACAUG 21 upstream (SEQ ID NO: 554) CEP290-485 +GGAUAGGACAGAGGACAUG (SEQ 19 upstream ID NO: 555) CEP290-484 +GAUAGGACAGAGGACAUG (SEQ ID 18 upstream NO: 556) CEP290-314 −GAAUAAAUGUAGAAUUUUAAUG 22 upstream (SEQ ID NO: 557) CEP290-64 −GUCAAAAGCUACCGGUUACCUG 22 downstream (SEQ ID NO: 558) CEP290-315 +GUUUUUAAGGCGGGGAGUCACAU 23 downstream (SEQ ID NO: 559) CEP290-203 −GUCUUACAUCCUCCUUACUGCCAC 24 downstream (SEQ ID NO: 560) CEP290-316 +GAGUCACAGGGUAGGAUUCAUGUU 24 downstream (SEQ ID NO: 561) CEP290-317 +GUCACAGGGUAGGAUUCAUGUU 22 downstream (SEQ ID NO: 562) CEP290-318 −GGCACAGAGUUCAAGCUAAUACAU 24 downstream (SEQ ID NO: 563) CEP290-319 −GCACAGAGUUCAAGCUAAUACAU 23 downstream (SEQ ID NO: 564) CEP290-505 −GAGUUCAAGCUAAUACAU (SEQ ID 18 downstream NO: 565) CEP290-496 +GAUGCAGAACUAGUGUAGAC (SEQ 20 downstream ID NO: 460) CEP290-320 −GUGUUGAGUAUCUCCUGUUUGGCA 24 downstream (SEQ ID NO: 566) CEP290-321 −GUUGAGUAUCUCCUGUUUGGCA 22 downstream (SEQ ID NO: 567) CEP290-504 −GAGUAUCUCCUGUUUGGCA (SEQ ID 19 downstream NO: 568) CEP290-322 −GAAAAUCAGAUUUCAUGUGUG 21 downstream (SEQ ID NO: 569) CEP290-324 −GCCACAAGAAUGAUCAUUCUAAAC 24 downstream (SEQ ID NO: 570) CEP290-325 +GGCGGGGAGUCACAUGGGAGUCA 23 downstream (SEQ ID NO: 571) CEP290-326 +GCGGGGAGUCACAUGGGAGUCA 22 downstream (SEQ ID NO: 572) CEP290-499 +GGGGAGUCACAUGGGAGUCA (SEQ 20 downstream ID NO: 573) CEP290-498 +GGGAGUCACAUGGGAGUCA (SEQ ID 19 downstream NO: 574) CEP290-497 +GGAGUCACAUGGGAGUCA (SEQ ID 18 downstream NO: 575) CEP290-327 +GCUUUUGACAGUUUUUAAGGCG 22 downstream (SEQ ID NO: 576) CEP290-328 +GAUCAUUCUUGUGGCAGUAAG 21 downstream (SEQ ID NO: 577) CEP290-329 −GAGCAAGAGAUGAACUAG (SEQ ID 18 downstream NO: 578) CEP290-500 +GCCUGAACAAGUUUUGAAAC (SEQ 20 downstream ID NO: 480) CEP290-330 −GUAGAUUGAGGUAGAAUCAAGAA 23 downstream (SEQ ID NO: 579) CEP290-506 −GAUUGAGGUAGAAUCAAGAA (SEQ 20 downstream ID NO: 580) CEP290-331 +GGAUGUAAGACUGGAGAUAGAGAC 24 downstream (SEQ ID NO: 581) CEP290-332 +GAUGUAAGACUGGAGAUAGAGAC 23 downstream (SEQ ID NO: 582) CEP290-503 +GUAAGACUGGAGAUAGAGAC (SEQ 20 downstream ID NO: 497) CEP290-333 +GGGAGUCACAUGGGAGUCACAGGG 24 downstream (SEQ ID NO: 583) CEP290-334 +GGAGUCACAUGGGAGUCACAGGG 23 downstream (SEQ ID NO: 584) CEP290-335 +GAGUCACAUGGGAGUCACAGGG 22 downstream (SEQ ID NO: 585) CEP290-502 +GUCACAUGGGAGUCACAGGG (SEQ 20 downstream ID NO: 586) CEP290-336 −GUUUACAUAUCUGUCUUCCUUAA 23 downstream (SEQ ID NO: 587) CEP290-507 −GAUUUCAUGUGUGAAGAA (SEQ ID 18 downstream NO: 588)

Table 9B provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the second tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation, andhave good orthogonality. It is contemplated herein that the targetingdomain hybridizes to the target domain through complementary basepairing. Any of the targeting domains in the table can be used with a S.aureus Cas9 molecule that generates a double stranded break (Cas9nuclease) or a single-stranded break (Cas9 nickase).

TABLE 9B Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-337 + AAAUCAUGCAAGUGACCUAAG 21 upstream(SEQ ID NO: 1191) CEP290-338 + AAUCAUGCAAGUGACCUAAG (SEQ 20 upstream IDNO: 1192) CEP290-339 + AUCAUGCAAGUGACCUAAG (SEQ ID 19 upstream NO: 1193)CEP290-340 − AGGUCACUUGCAUGAUUUAG (SEQ 20 upstream ID NO: 1194)CEP290-341 − AAUAUUAAGGGCUCUUCCUGGACC 24 upstream (SEQ ID NO: 1195)CEP290-342 − AUAUUAAGGGCUCUUCCUGGACC 23 upstream (SEQ ID NO: 1196)CEP290-343 − AUUAAGGGCUCUUCCUGGACC (SEQ 21 upstream ID NO: 1197)CEP290-344 − AAGGGCUCUUCCUGGACC (SEQ ID 18 upstream NO: 1198)CEP290-345 + AGGACUUUCUAAUGCUGGA (SEQ ID 19 upstream NO: 1199)CEP290-346 + ACCAUGGGAGAAUAGUUUGUU 21 upstream (SEQ ID NO: 1200)CEP290-347 + AUGGGAGAAUAGUUUGUU (SEQ ID 18 upstream NO: 1201)CEP290-348 + ACUCCUGUGAAAGGAUCUUAGAU 23 upstream (SEQ ID NO: 1202)CEP290-349 − AAAACGUUGUUCUGAGUAGCUUU 23 upstream (SEQ ID NO: 1203)CEP290-350 − AAACGUUGUUCUGAGUAGCUUU 22 upstream (SEQ ID NO: 1204)CEP290-351 − AACGUUGUUCUGAGUAGCUUU 21 upstream (SEQ ID NO: 1205)CEP290-352 − ACGUUGUUCUGAGUAGCUUU (SEQ 20 upstream ID NO: 749)CEP290-353 − AUUUAUAGUGGCUGAAUGACUU 22 upstream (SEQ ID NO: 1207)CEP290-354 − AUAGUGGCUGAAUGACUU (SEQ ID 18 upstream NO: 1208) CEP290-355− AUGGUCCCUGGCUUUUGUUCCU 22 upstream (SEQ ID NO: 1209) CEP290-356 −AGACAUCUUGUGGAUAAUGUAUCA 24 upstream (SEQ ID NO: 1210) CEP290-357 −ACAUCUUGUGGAUAAUGUAUCA 22 upstream (SEQ ID NO: 1211) CEP290-358 −AUCUUGUGGAUAAUGUAUCA (SEQ 20 upstream ID NO: 835) CEP290-359 −AAAGUCCUAGGCAAGAGACAUCUU 24 upstream (SEQ ID NO: 1213) CEP290-360 −AAGUCCUAGGCAAGAGACAUCUU 23 upstream (SEQ ID NO: 1214) CEP290-361 −AGUCCUAGGCAAGAGACAUCUU 22 upstream (SEQ ID NO: 1215) CEP290-362 +AGCCAGCAAAAGCUUUUGAGCUAA 24 upstream (SEQ ID NO: 1216) CEP290-363 +AGCAAAAGCUUUUGAGCUAA (SEQ 20 upstream ID NO: 763) CEP290-364 +AGAUCUUAUUCUACUCCUGUGA 22 upstream (SEQ ID NO: 1218) CEP290-365 +AUCUUAUUCUACUCCUGUGA (SEQ 20 upstream ID NO: 764) CEP290-366 −AUCUAAGAUCCUUUCACAGGAG 22 upstream (SEQ ID NO: 1220) CEP290-369 −AAGAUCCUUUCACAGGAG (SEQ ID 18 upstream NO: 1221) CEP290-370 −AGCUUUCAGGAUUCCUACUAAAUU 24 upstream (SEQ ID NO: 1222) CEP290-371 +ACUCAGAACAACGUUUUCAUUUA 23 upstream (SEQ ID NO: 1223) CEP290-372 +AGAACAACGUUUUCAUUUA (SEQ ID 19 upstream NO: 1224) CEP290-373 −AGAAUAUCAUAAGUUACAAUCU 22 upstream (SEQ ID NO: 1225) CEP290-375 −AAUAUCAUAAGUUACAAUCU (SEQ 20 upstream ID NO: 1226) CEP290-376 −AUAUCAUAAGUUACAAUCU (SEQ ID 19 upstream NO: 1227) CEP290-377 +AAGUGGCUGUAAGAUAACUACA 22 upstream (SEQ ID NO: 1228) CEP290-378 +AGUGGCUGUAAGAUAACUACA 21 upstream (SEQ ID NO: 1229) CEP290-379 −AUGUUUAACGUUAUCAUUUUCCCA 24 upstream (SEQ ID NO: 1230) CEP290-380 −AACGUUAUCAUUUUCCCA (SEQ ID 18 upstream NO: 1231) CEP290-381 +AAGAGAAAGGGAUGGGCACUUA 22 upstream (SEQ ID NO: 1232) CEP290-382 +AGAGAAAGGGAUGGGCACUUA 21 upstream (SEQ ID NO: 1233) CEP290-383 +AGAAAGGGAUGGGCACUUA (SEQ 19 upstream ID NO: 1234) CEP290-384 −AUUCAGUAAAUGAAAACGUUGUU 23 upstream (SEQ ID NO: 1235) CEP290-385 −AGUAAAUGAAAACGUUGUU (SEQ 19 upstream ID NO: 1236) CEP290-386 +AUAAACAUGACUCAUAAUUUAGU 23 upstream (SEQ ID NO: 1237) CEP290-387 +AAACAUGACUCAUAAUUUAGU 21 upstream (SEQ ID NO: 1238) CEP290-388 +AACAUGACUCAUAAUUUAGU (SEQ 20 upstream ID NO: 721) CEP290-389 +ACAUGACUCAUAAUUUAGU (SEQ ID 19 upstream NO: 1240) CEP290-390 −AUUCUUAUCUAAGAUCCUUUCAC 23 upstream (SEQ ID NO: 1241) CEP290-391 +AGGAACAAAAGCCAGGGACCAUGG 24 upstream (SEQ ID NO: 1242) CEP290-392 +AACAAAAGCCAGGGACCAUGG 21 upstream (SEQ ID NO: 1243) CEP290-393 +ACAAAAGCCAGGGACCAUGG (SEQ 20 upstream ID NO: 1244) CEP290-394 +AAAAGCCAGGGACCAUGG (SEQ ID 18 upstream NO: 1245) CEP290-395 +AGAAUAGUUUGUUCUGGGUAC 21 upstream (SEQ ID NO: 1246) CEP290-396 +AAUAGUUUGUUCUGGGUAC (SEQ 19 upstream ID NO: 1247) CEP290-397 +AUAGUUUGUUCUGGGUAC (SEQ ID 18 upstream NO: 1248) CEP290-398 −AUCAGAAAUAGAGGCUUAUGGAUU 24 upstream (SEQ ID NO: 1249) CEP290-399 −AGAAAUAGAGGCUUAUGGAUU 21 upstream (SEQ ID NO: 1250) CEP290-400 −AAAUAGAGGCUUAUGGAUU (SEQ 19 upstream ID NO: 1251) CEP290-401 −AAUAGAGGCUUAUGGAUU (SEQ ID 18 upstream NO: 1252) CEP290-402 −AAUAUAUUAUCUAUUUAUAGUGG 23 upstream (SEQ ID NO: 1253) CEP290-403 −AUAUAUUAUCUAUUUAUAGUGG 22 upstream (SEQ ID NO: 1254) CEP290-404 −AUAUUAUCUAUUUAUAGUGG (SEQ 20 upstream ID NO: 1255) CEP290-405 −AUUAUCUAUUUAUAGUGG (SEQ ID 18 upstream NO: 1256) CEP290-406 −AAAUUCUCAUCAUUUUUUAUUG 22 upstream (SEQ ID NO: 1257) CEP290-407 −AAUUCUCAUCAUUUUUUAUUG 21 upstream (SEQ ID NO: 1258) CEP290-408 −AUUCUCAUCAUUUUUUAUUG (SEQ 20 upstream ID NO: 1259) CEP290-409 +AGAGGAUAGGACAGAGGACAUG 22 upstream (SEQ ID NO: 1260) CEP290-410 +AGGAUAGGACAGAGGACAUG (SEQ 20 upstream ID NO: 827) CEP290-411 −AGAAUAAAUGUAGAAUUUUAAUG 23 upstream (SEQ ID NO: 1262) CEP290-412 −AAUAAAUGUAGAAUUUUAAUG 21 upstream (SEQ ID NO: 1263) CEP290-413 −AUAAAUGUAGAAUUUUAAUG (SEQ 20 upstream ID NO: 1264) CEP290-414 −AAAUGUAGAAUUUUAAUG (SEQ ID 18 upstream NO: 1265) CEP290-415 −AUUUUUUAUUGUAGAAUAAAUG 22 upstream (SEQ ID NO: 1266) CEP290-416 +CUAAAUCAUGCAAGUGACCUAAG 23 upstream (SEQ ID NO: 1267) CEP290-417 −CCUUAGGUCACUUGCAUGAUUUAG 24 upstream (SEQ ID NO: 1268) CEP290-418 −CUUAGGUCACUUGCAUGAUUUAG 23 upstream (SEQ ID NO: 1269) CEP290-419 +CCUAGGACUUUCUAAUGCUGGA 22 upstream (SEQ ID NO: 1270) CEP290-420 +CUAGGACUUUCUAAUGCUGGA 21 upstream (SEQ ID NO: 1271) CEP290-421 +CCAUGGGAGAAUAGUUUGUU (SEQ 20 upstream ID NO: 1272) CEP290-422 +CAUGGGAGAAUAGUUUGUU (SEQ 19 upstream ID NO: 1273) CEP290-423 +CUCCUGUGAAAGGAUCUUAGAU 22 upstream (SEQ ID NO: 1274) CEP290-424 +CCUGUGAAAGGAUCUUAGAU (SEQ 20 upstream ID NO: 860) CEP290-426 +CUGUGAAAGGAUCUUAGAU (SEQ 19 upstream ID NO: 1276) CEP290-427 −CCCUGGCUUUUGUUCCUUGGA (SEQ 21 upstream ID NO: 1277) CEP290-428 −CCUGGCUUUUGUUCCUUGGA (SEQ 20 upstream ID NO: 1278) CEP290-429 −CUGGCUUUUGUUCCUUGGA (SEQ ID 19 upstream NO: 1279) CEP290-430 −CGUUGUUCUGAGUAGCUUU (SEQ ID 19 upstream NO: 1280) CEP290-431 −CUAUUUAUAGUGGCUGAAUGACUU 24 upstream (SEQ ID NO: 1281) CEP290-432 −CCAUGGUCCCUGGCUUUUGUUCCU 24 upstream (SEQ ID NO: 1282) CEP290-433 −CAUGGUCCCUGGCUUUUGUUCCU 23 upstream (SEQ ID NO: 1283) CEP290-434 −CAUCUUGUGGAUAAUGUAUCA 21 upstream (SEQ ID NO: 1284) CEP290-435 −CUUGUGGAUAAUGUAUCA (SEQ ID 18 upstream NO: 1285) CEP290-437 −CCUAGGCAAGAGACAUCUU (SEQ ID 19 upstream NO: 1286) CEP290-438 −CUAGGCAAGAGACAUCUU (SEQ ID 18 upstream NO: 1287) CEP290-439 +CCAGCAAAAGCUUUUGAGCUAA 22 upstream (SEQ ID NO: 1288) CEP290-440 +CAGCAAAAGCUUUUGAGCUAA 21 upstream (SEQ ID NO: 1289) CEP290-441 +CAAAAGCUUUUGAGCUAA (SEQ ID 18 upstream NO: 1290) CEP290-442 +CUUAUUCUACUCCUGUGA (SEQ ID 18 upstream NO: 1291) CEP290-443 −CUAAGAUCCUUUCACAGGAG (SEQ 20 upstream ID NO: 861) CEP290-444 −CUUCCUCAUCAGAAAUAGAGGCUU 24 upstream (SEQ ID NO: 1293) CEP290-445 −CCUCAUCAGAAAUAGAGGCUU 21 upstream (SEQ ID NO: 1294) CEP290-446 −CUCAUCAGAAAUAGAGGCUU (SEQ 20 upstream ID NO: 865) CEP290-447 −CAUCAGAAAUAGAGGCUU (SEQ ID 18 upstream NO: 1296) CEP290-448 −CUUUCAGGAUUCCUACUAAAUU 22 upstream (SEQ ID NO: 1297) CEP290-449 −CAGGAUUCCUACUAAAUU (SEQ ID 18 upstream NO: 1298) CEP290-450 +CUGUCCUCAGUAAAAGGUA (SEQ ID 19 upstream NO: 1299) CEP290-451 +CUCAGAACAACGUUUUCAUUUA 22 upstream (SEQ ID NO: 1300) CEP290-452 +CAGAACAACGUUUUCAUUUA (SEQ 20 upstream ID NO: 761) CEP290-453 +CAAGUGGCUGUAAGAUAACUACA 23 upstream (SEQ ID NO: 1302) CEP290-454 −CAUUCAGUAAAUGAAAACGUUGUU 24 upstream (SEQ ID NO: 1303) CEP290-457 −CAGUAAAUGAAAACGUUGUU (SEQ 20 upstream ID NO: 849) CEP290-458 +CAUGACUCAUAAUUUAGU (SEQ ID 18 upstream NO: 1305) CEP290-459 −CUUAUCUAAGAUCCUUUCAC (SEQ 20 upstream ID NO: 734) CEP290-460 +CAAAAGCCAGGGACCAUGG (SEQ ID 19 upstream NO: 1307) CEP290-461 −CAGAAAUAGAGGCUUAUGGAUU 22 upstream (SEQ ID NO: 1308) CEP290-462 +CUGGGUACAGGGGUAAGAGAA 21 upstream (SEQ ID NO: 1309) CEP290-463 −CAAUAUAUUAUCUAUUUAUAGUGG 24 upstream (SEQ ID NO: 1310) CEP290-464 −CAUUUUUUAUUGUAGAAUAAAUG 23 upstream (SEQ ID NO: 1311) CEP290-465 +UAAAUCAUGCAAGUGACCUAAG 22 upstream (SEQ ID NO: 1312) CEP290-466 +UCAUGCAAGUGACCUAAG (SEQ ID 18 upstream NO: 1313) CEP290-467 −UUAGGUCACUUGCAUGAUUUAG 22 upstream (SEQ ID NO: 1314) CEP290-468 −UAGGUCACUUGCAUGAUUUAG 21 upstream (SEQ ID NO: 1315) CEP290-469 −UAUUAAGGGCUCUUCCUGGACC 22 upstream (SEQ ID NO: 1316) CEP290-470 −UUAAGGGCUCUUCCUGGACC (SEQ 20 upstream ID NO: 1317) CEP290-471 −UAAGGGCUCUUCCUGGACC (SEQ ID 19 upstream NO: 1318) CEP290-472 +UGCCUAGGACUUUCUAAUGCUGGA 24 upstream (SEQ ID NO: 1319) CEP290-473 +UAGGACUUUCUAAUGCUGGA (SEQ 20 upstream ID NO: 882) CEP290-474 +UACUCCUGUGAAAGGAUCUUAGAU 24 upstream (SEQ ID NO: 1321) CEP290-475 +UCCUGUGAAAGGAUCUUAGAU 21 upstream (SEQ ID NO: 1322) CEP290-476 +UGUGAAAGGAUCUUAGAU (SEQ ID 18 upstream NO: 1323) CEP290-477 −UCCCUGGCUUUUGUUCCUUGGA 22 upstream (SEQ ID NO: 1324) CEP290-515 −UGGCUUUUGUUCCUUGGA (SEQ ID 18 upstream NO: 1325) CEP290-516 −UAUUUAUAGUGGCUGAAUGACUU 23 upstream (SEQ ID NO: 1326) CEP290-517 −UUUAUAGUGGCUGAAUGACUU 21 upstream (SEQ ID NO: 1327) CEP290-518 −UUAUAGUGGCUGAAUGACUU (SEQ 20 upstream ID NO: 1328) CEP290-519 −UAUAGUGGCUGAAUGACUU (SEQ 19 upstream ID NO: 1329) CEP290-520 −UGGUCCCUGGCUUUUGUUCCU (SEQ 21 upstream ID NO: 1330) CEP290-521 −UCCCUGGCUUUUGUUCCU (SEQ ID 18 upstream NO: 1331) CEP290-522 −UCUUGUGGAUAAUGUAUCA (SEQ 19 upstream ID NO: 1332) CEP290-523 −UCCUAGGCAAGAGACAUCUU (SEQ 20 upstream ID NO: 887) CEP290-524 +UUAGAUCUUAUUCUACUCCUGUGA 24 upstream (SEQ ID NO: 1334) CEP290-525 +UAGAUCUUAUUCUACUCCUGUGA 23 upstream (SEQ ID NO: 1335) CEP290-526 +UCUUAUUCUACUCCUGUGA (SEQ ID 19 upstream NO: 1336) CEP290-527 −UUAUCUAAGAUCCUUUCACAGGAG 24 upstream (SEQ ID NO: 1337) CEP290-528 −UAUCUAAGAUCCUUUCACAGGAG 23 upstream (SEQ ID NO: 1338) CEP290-529 −UCUAAGAUCCUUUCACAGGAG 21 upstream (SEQ ID NO: 1339) CEP290-530 −UAAGAUCCUUUCACAGGAG (SEQ ID 19 upstream NO: 1340) CEP290-531 −UUCCUCAUCAGAAAUAGAGGCUU 23 upstream (SEQ ID NO: 1341) CEP290-532 −UCCUCAUCAGAAAUAGAGGCUU 22 upstream (SEQ ID NO: 1342) CEP290-533 −UCAUCAGAAAUAGAGGCUU (SEQ ID 19 upstream NO: 1343) CEP290-534 −UUUCAGGAUUCCUACUAAAUU 21 upstream (SEQ ID NO: 1344) CEP290-535 −UUCAGGAUUCCUACUAAAUU (SEQ 20 upstream ID NO: 904) CEP290-536 −UCAGGAUUCCUACUAAAUU (SEQ ID 19 upstream NO: 1346) CEP290-537 +UUGUUCUGUCCUCAGUAAAAGGUA 24 upstream (SEQ ID NO: 1347) CEP290-538 +UGUUCUGUCCUCAGUAAAAGGUA 23 upstream (SEQ ID NO: 1348) CEP290-539 +UUCUGUCCUCAGUAAAAGGUA 21 upstream (SEQ ID NO: 1349) CEP290-540 +UCUGUCCUCAGUAAAAGGUA (SEQ 20 upstream ID NO: 890) CEP290-541 +UGUCCUCAGUAAAAGGUA (SEQ ID 18 upstream NO: 1351) CEP290-542 +UACUCAGAACAACGUUUUCAUUUA 24 upstream (SEQ ID NO: 1352) CEP290-543 +UCAGAACAACGUUUUCAUUUA 21 upstream (SEQ ID NO: 1353) CEP290-544 −UAGAAUAUCAUAAGUUACAAUCU 23 upstream (SEQ ID NO: 1354) CEP290-545 −UAUCAUAAGUUACAAUCU (SEQ ID 18 upstream NO: 1355) CEP290-546 +UCAAGUGGCUGUAAGAUAACUACA 24 upstream (SEQ ID NO: 1356) CEP290-547 +UGGCUGUAAGAUAACUACA (SEQ ID 19 upstream NO: 1357) CEP290-548 −UGUUUAACGUUAUCAUUUUCCCA 23 upstream (SEQ ID NO: 1358) CEP290-549 −UUUAACGUUAUCAUUUUCCCA 21 upstream (SEQ ID NO: 1359) CEP290-550 −UUAACGUUAUCAUUUUCCCA (SEQ 20 upstream ID NO: 900) CEP290-551 −UAACGUUAUCAUUUUCCCA (SEQ ID 19 upstream NO: 1361) CEP290-552 +UAAGAGAAAGGGAUGGGCACUUA 23 upstream (SEQ ID NO: 1362) CEP290-553 −UUCAGUAAAUGAAAACGUUGUU 22 upstream (SEQ ID NO: 1363) CEP290-554 −UCAGUAAAUGAAAACGUUGUU 21 upstream (SEQ ID NO: 1364) CEP290-555 +UAAACAUGACUCAUAAUUUAGU 22 upstream (SEQ ID NO: 1365) CEP290-556 −UAUUCUUAUCUAAGAUCCUUUCAC 24 upstream (SEQ ID NO: 1366) CEP290-557 −UUCUUAUCUAAGAUCCUUUCAC 22 upstream (SEQ ID NO: 1367) CEP290-558 −UCUUAUCUAAGAUCCUUUCAC (SEQ 21 upstream ID NO: 1368) CEP290-559 −UUAUCUAAGAUCCUUUCAC (SEQ ID 19 upstream NO: 1369) CEP290-560 −UAUCUAAGAUCCUUUCAC (SEQ ID 18 upstream NO: 1370) CEP290-561 −UCAGAAAUAGAGGCUUAUGGAUU 23 upstream (SEQ ID NO: 1371) CEP290-562 +UUCUGGGUACAGGGGUAAGAGAA 23 upstream (SEQ ID NO: 1372) CEP290-563 +UCUGGGUACAGGGGUAAGAGAA 22 upstream (SEQ ID NO: 1373) CEP290-564 +UGGGUACAGGGGUAAGAGAA (SEQ 20 upstream ID NO: 1374) CEP290-565 −UAUAUUAUCUAUUUAUAGUGG 21 upstream (SEQ ID NO: 1375) CEP290-566 −UAUUAUCUAUUUAUAGUGG (SEQ 19 upstream ID NO: 1376) CEP290-567 −UAAAUUCUCAUCAUUUUUUAUUG 23 upstream (SEQ ID NO: 1377) CEP290-568 −UUCUCAUCAUUUUUUAUUG (SEQ ID 19 upstream NO: 1378) CEP290-569 −UCUCAUCAUUUUUUAUUG (SEQ ID 18 upstream NO: 1379) CEP290-570 −UAGAAUAAAUGUAGAAUUUUAAUG 24 upstream (SEQ ID NO: 1380) CEP290-571 −UAAAUGUAGAAUUUUAAUG (SEQ 19 upstream ID NO: 1381) CEP290-572 −UCAUUUUUUAUUGUAGAAUAAAUG 24 upstream (SEQ ID NO: 1382) CEP290-573 −UUUUUUAUUGUAGAAUAAAUG 21 upstream (SEQ ID NO: 1383) CEP290-574 −UUUUUAUUGUAGAAUAAAUG (SEQ 20 upstream ID NO: 1384) CEP290-575 −UUUUAUUGUAGAAUAAAUG (SEQ 19 upstream ID NO: 1385) CEP290-576 −UUUAUUGUAGAAUAAAUG (SEQ ID 18 upstream NO: 1386) CEP290-577 −AAAAGCUACCGGUUACCUG (SEQ ID 19 downstream NO: 1387) CEP290-578 −AAAGCUACCGGUUACCUG (SEQ ID 18 downstream NO: 1388) CEP290-579 +AGUUUUUAAGGCGGGGAGUCACAU 24 downstream (SEQ ID NO: 1389) CEP290-580 −ACAUCCUCCUUACUGCCAC (SEQ ID 19 downstream NO: 1390) CEP290-581 +AGUCACAGGGUAGGAUUCAUGUU 23 downstream (SEQ ID NO: 1391) CEP290-582 +ACAGGGUAGGAUUCAUGUU (SEQ 19 downstream ID NO: 1392) CEP290-583 −ACAGAGUUCAAGCUAAUACAU 21 downstream (SEQ ID NO: 1393) CEP290-584 −AGAGUUCAAGCUAAUACAU (SEQ ID 19 downstream NO: 1394) CEP290-585 +AUAAGAUGCAGAACUAGUGUAGAC 24 downstream (SEQ ID NO: 1395) CEP290-586 +AAGAUGCAGAACUAGUGUAGAC 22 downstream (SEQ ID NO: 1396) CEP290-587 +AGAUGCAGAACUAGUGUAGAC 21 downstream (SEQ ID NO: 1397) CEP290-588 +AUGCAGAACUAGUGUAGAC (SEQ ID 19 downstream NO: 1398) CEP290-589 −AGUAUCUCCUGUUUGGCA (SEQ ID 18 downstream NO: 1399) CEP290-590 −ACGAAAAUCAGAUUUCAUGUGUG 23 downstream (SEQ ID NO: 1400) CEP290-591 −AAAAUCAGAUUUCAUGUGUG (SEQ 20 downstream ID NO: 1401) CEP290-592 −AAAUCAGAUUUCAUGUGUG (SEQ 19 downstream ID NO: 1402) CEP290-593 −AAUCAGAUUUCAUGUGUG (SEQ ID 18 downstream NO: 1403) CEP290-594 −ACAAGAAUGAUCAUUCUAAAC 21 downstream (SEQ ID NO: 1404) CEP290-595 −AAGAAUGAUCAUUCUAAAC (SEQ ID 19 downstream NO: 1405) CEP290-596 −AGAAUGAUCAUUCUAAAC (SEQ ID 18 downstream NO: 1406) CEP290-597 +AGGCGGGGAGUCACAUGGGAGUCA 24 downstream (SEQ ID NO: 1407) CEP290-598 +AGCUUUUGACAGUUUUUAAGGCG 23 downstream (SEQ ID NO: 1408) CEP290-599 +AAUGAUCAUUCUUGUGGCAGUAAG 24 downstream (SEQ ID NO: 1409) CEP290-600 +AUGAUCAUUCUUGUGGCAGUAAG 23 downstream (SEQ ID NO: 1410) CEP290-601 +AUCAUUCUUGUGGCAGUAAG (SEQ 20 downstream ID NO: 833) CEP290-602 −AUCUAGAGCAAGAGAUGAACUAG 23 downstream (SEQ ID NO: 1412) CEP290-603 −AGAGCAAGAGAUGAACUAG (SEQ 19 downstream ID NO: 1413) CEP290-604 +AAUGCCUGAACAAGUUUUGAAAC 23 downstream (SEQ ID NO: 1414) CEP290-605 +AUGCCUGAACAAGUUUUGAAAC 22 downstream (SEQ ID NO: 1415) CEP290-606 −AGAUUGAGGUAGAAUCAAGAA 21 downstream (SEQ ID NO: 1416) CEP290-607 −AUUGAGGUAGAAUCAAGAA (SEQ 19 downstream ID NO: 1417) CEP290-608 +AUGUAAGACUGGAGAUAGAGAC 22 downstream (SEQ ID NO: 1418) CEP290-609 +AAGACUGGAGAUAGAGAC (SEQ ID 18 downstream NO: 1419) CEP290-610 +AGUCACAUGGGAGUCACAGGG 21 downstream (SEQ ID NO: 1420) CEP290-611 −ACAUAUCUGUCUUCCUUAA (SEQ ID 19 downstream NO: 1421) CEP290-612 −AAAUCAGAUUUCAUGUGUGAAGAA 24 downstream (SEQ ID NO: 1422) CEP290-613 −AAUCAGAUUUCAUGUGUGAAGAA 23 downstream (SEQ ID NO: 1423) CEP290-614 −AUCAGAUUUCAUGUGUGAAGAA 22 downstream (SEQ ID NO: 1424) CEP290-615 −AGAUUUCAUGUGUGAAGAA (SEQ 19 downstream ID NO: 1425) CEP290-616 +AAAUAAAACUAAGACACUGCCAAU 24 downstream (SEQ ID NO: 1025) CEP290-617 +AAUAAAACUAAGACACUGCCAAU 23 downstream (SEQ ID NO: 1026) CEP290-618 +AUAAAACUAAGACACUGCCAAU 22 downstream (SEQ ID NO: 1027) CEP290-619 +AAAACUAAGACACUGCCAAU (SEQ 20 downstream ID NO: 610) CEP290-620 +AAACUAAGACACUGCCAAU (SEQ ID 19 downstream NO: 1028) CEP290-621 +AACUAAGACACUGCCAAU (SEQ ID 18 downstream NO: 1029) CEP290-622 −AACUAUUUAAUUUGUUUCUGUGUG 24 downstream (SEQ ID NO: 1431) CEP290-623 −ACUAUUUAAUUUGUUUCUGUGUG 23 downstream (SEQ ID NO: 1432) CEP290-624 −AUUUAAUUUGUUUCUGUGUG (SEQ 20 downstream ID NO: 840) CEP290-625 −CUGUCAAAAGCUACCGGUUACCUG 24 downstream (SEQ ID NO: 1434) CEP290-626 −CAAAAGCUACCGGUUACCUG (SEQ 20 downstream ID NO: 755) CEP290-627 −CUUACAUCCUCCUUACUGCCAC 22 downstream (SEQ ID NO: 1436) CEP290-628 −CAUCCUCCUUACUGCCAC (SEQ ID 18 downstream NO: 1437) CEP290-629 +CACAGGGUAGGAUUCAUGUU (SEQ 20 downstream ID NO: 846) CEP290-630 +CAGGGUAGGAUUCAUGUU (SEQ ID 18 downstream NO: 1439) CEP290-631 −CACAGAGUUCAAGCUAAUACAU 22 downstream (SEQ ID NO: 1440) CEP290-632 −CAGAGUUCAAGCUAAUACAU (SEQ 20 downstream ID NO: 848) CEP290-633 −CACGAAAAUCAGAUUUCAUGUGUG 24 downstream (SEQ ID NO: 1442) CEP290-634 −CGAAAAUCAGAUUUCAUGUGUG 22 downstream (SEQ ID NO: 1443) CEP290-635 −CCACAAGAAUGAUCAUUCUAAAC 23 downstream (SEQ ID NO: 1444) CEP290-636 −CACAAGAAUGAUCAUUCUAAAC 22 downstream (SEQ ID NO: 1445) CEP290-637 −CAAGAAUGAUCAUUCUAAAC (SEQ 20 downstream ID NO: 844) CEP290-638 +CGGGGAGUCACAUGGGAGUCA 21 downstream (SEQ ID NO: 1447) CEP290-639 +CUUUUGACAGUUUUUAAGGCG 21 downstream (SEQ ID NO: 1448) CEP290-640 +CAUUCUUGUGGCAGUAAG (SEQ ID 18 downstream NO: 1449) CEP290-641 −CAUCUAGAGCAAGAGAUGAACUAG 24 downstream (SEQ ID NO: 1450) CEP290-642 −CUAGAGCAAGAGAUGAACUAG 21 downstream (SEQ ID NO: 1451) CEP290-643 +CCUGAACAAGUUUUGAAAC (SEQ ID 19 downstream NO: 1452) CEP290-644 +CUGAACAAGUUUUGAAAC (SEQ ID 18 downstream NO: 1453) CEP290-645 −CUCUCUUCCAGUUGUUUUGCUCA 23 downstream (SEQ ID NO: 1454) CEP290-646 −CUCUUCCAGUUGUUUUGCUCA (SEQ 21 downstream ID NO: 1455) CEP290-647 −CUUCCAGUUGUUUUGCUCA (SEQ ID 19 downstream NO: 1456) CEP290-648 +CACAUGGGAGUCACAGGG (SEQ ID 18 downstream NO: 1457) CEP290-649 −CAUAUCUGUCUUCCUUAA (SEQ ID 18 downstream NO: 1458) CEP290-650 −CAGAUUUCAUGUGUGAAGAA (SEQ 20 downstream ID NO: 1124) CEP290-651 −CUAUUUAAUUUGUUUCUGUGUG 22 downstream (SEQ ID NO: 1460) CEP290-652 −UGUCAAAAGCUACCGGUUACCUG 23 downstream (SEQ ID NO: 1461) CEP290-653 −UCAAAAGCUACCGGUUACCUG (SEQ 21 downstream ID NO: 1462) CEP290-654 +UUUUUAAGGCGGGGAGUCACAU 22 downstream (SEQ ID NO: 1463) CEP290-655 +UUUUAAGGCGGGGAGUCACAU 21 downstream (SEQ ID NO: 1464) CEP290-656 +UUUAAGGCGGGGAGUCACAU (SEQ 20 downstream ID NO: 619) CEP290-657 +UUAAGGCGGGGAGUCACAU (SEQ ID 19 downstream NO: 1466) CEP290-658 +UAAGGCGGGGAGUCACAU (SEQ ID 18 downstream NO: 1467) CEP290-659 −UCUUACAUCCUCCUUACUGCCAC 23 downstream (SEQ ID NO: 1468) CEP290-660 −UUACAUCCUCCUUACUGCCAC (SEQ 21 downstream ID NO: 1469) CEP290-661 −UACAUCCUCCUUACUGCCAC (SEQ 20 downstream ID NO: 875) CEP290-662 +UCACAGGGUAGGAUUCAUGUU 21 downstream (SEQ ID NO: 1471) CEP290-663 +UAAGAUGCAGAACUAGUGUAGAC 23 downstream (SEQ ID NO: 1472) CEP290-664 +UGCAGAACUAGUGUAGAC (SEQ ID 18 downstream NO: 1473) CEP290-665 −UGUUGAGUAUCUCCUGUUUGGCA 23 downstream (SEQ ID NO: 1474) CEP290-666 −UUGAGUAUCUCCUGUUUGGCA 21 downstream (SEQ ID NO: 1475) CEP290-667 −UGAGUAUCUCCUGUUUGGCA (SEQ 20 downstream ID NO: 895) CEP290-668 +UAGCUUUUGACAGUUUUUAAGGCG 24 downstream (SEQ ID NO: 1477) CEP290-669 +UUUUGACAGUUUUUAAGGCG (SEQ 20 downstream ID NO: 681) CEP290-670 +UUUGACAGUUUUUAAGGCG (SEQ 19 downstream ID NO: 1479) CEP290-671 +UUGACAGUUUUUAAGGCG (SEQ ID 18 downstream NO: 1480) CEP290-672 +UGAUCAUUCUUGUGGCAGUAAG 22 downstream (SEQ ID NO: 1481) CEP290-673 +UCAUUCUUGUGGCAGUAAG (SEQ ID 19 downstream NO: 1482) CEP290-674 −UCUAGAGCAAGAGAUGAACUAG 22 downstream (SEQ ID NO: 1483) CEP290-675 −UAGAGCAAGAGAUGAACUAG (SEQ 20 downstream ID NO: 878) CEP290-676 +UAAUGCCUGAACAAGUUUUGAAAC 24 downstream (SEQ ID NO: 1485) CEP290-677 +UGCCUGAACAAGUUUUGAAAC 21 downstream (SEQ ID NO: 1486) CEP290-678 −UGUAGAUUGAGGUAGAAUCAAGAA 24 downstream (SEQ ID NO: 1487) CEP290-679 −UAGAUUGAGGUAGAAUCAAGAA 22 downstream (SEQ ID NO: 1488) CEP290-680 −UUGAGGUAGAAUCAAGAA (SEQ ID 18 downstream NO: 1489) CEP290-681 +UGUAAGACUGGAGAUAGAGAC 21 downstream (SEQ ID NO: 1490) CEP290-682 +UAAGACUGGAGAUAGAGAC (SEQ 19 downstream ID NO: 1491) CEP290-683 −UCUCUCUUCCAGUUGUUUUGCUCA 24 downstream (SEQ ID NO: 1492) CEP290-684 −UCUCUUCCAGUUGUUUUGCUCA 22 downstream (SEQ ID NO: 1493) CEP290-685 −UCUUCCAGUUGUUUUGCUCA (SEQ 20 downstream ID NO: 893) CEP290-686 −UUCCAGUUGUUUUGCUCA (SEQ ID 18 downstream NO: 1495) CEP290-687 +UCACAUGGGAGUCACAGGG (SEQ ID 19 downstream NO: 1496) CEP290-688 −UGUUUACAUAUCUGUCUUCCUUAA 24 downstream (SEQ ID NO: 1497) CEP290-689 −UUUACAUAUCUGUCUUCCUUAA 22 downstream (SEQ ID NO: 1498) CEP290-690 −UUACAUAUCUGUCUUCCUUAA 21 downstream (SEQ ID NO: 1499) CEP290-691 −UACAUAUCUGUCUUCCUUAA (SEQ 20 downstream ID NO: 689) CEP290-692 −UCAGAUUUCAUGUGUGAAGAA 21 downstream (SEQ ID NO: 1501) CEP290-693 +UAAAACUAAGACACUGCCAAU 21 downstream (SEQ ID NO: 1035) CEP290-694 −UAUUUAAUUUGUUUCUGUGUG 21 downstream (SEQ ID NO: 1503) CEP290-695 −UUUAAUUUGUUUCUGUGUG (SEQ 19 downstream ID NO: 1504) CEP290-696 −UUAAUUUGUUUCUGUGUG (SEQ ID 18 downstream NO: 1505)

Table 9C provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the third tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation, startwith G and PAM is NNGRRT. It is contemplated herein that the targetingdomain hybridizes to the target domain through complementary basepairing. Any of the targeting domains in the table can be used with a S.aureus Cas9 molecule that generates a double stranded break (Cas9nuclease) or a single-stranded break (Cas9 nickase).

TABLE 9C Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-697 − GUAGAAUAAAUUUAUUUAAUG 21 upstream(SEQ ID NO: 1506) CEP290-495 − GAAUAAAUUUAUUUAAUG (SEQ ID 18 upstreamNO: 1507) CEP290-698 − GAGAAAAAGGAGCAUGAAACAGG 23 upstream (SEQ ID NO:1508) CEP290-699 − GAAAAAGGAGCAUGAAACAGG 21 upstream (SEQ ID NO: 1509)CEP290-700 − GUAGAAUAAAAAAUAAAAAAAC 22 upstream (SEQ ID NO: 1510)CEP290-701 − GAAUAAAAAAUAAAAAAAC (SEQ 19 upstream ID NO: 1511)CEP290-702 − GAAUAAAAAAUAAAAAAACUAGAG 24 upstream (SEQ ID NO: 1512)CEP290-508 − GAAAUAGAUGUAGAUUGAGG (SEQ 20 downstream ID NO: 1513)CEP290-703 − GAUAAUAAGGAAAUACAAAAA 21 downstream (SEQ ID NO: 1514)CEP290-704 − GUGUUGCCCAGGCUGGAGUGCAG 23 downstream (SEQ ID NO: 1515)CEP290-705 − GUUGCCCAGGCUGGAGUGCAG 21 downstream (SEQ ID NO: 1516)CEP290-706 − GCCCAGGCUGGAGUGCAG (SEQ ID 18 downstream NO: 1517)CEP290-707 − GUUGUUUUUUUUUUUGAAA (SEQ 19 downstream ID NO: 1518)CEP290-708 − GAGUCUCACUGUGUUGCCCAGGC 23 downstream (SEQ ID NO: 1519)CEP290-709 − GUCUCACUGUGUUGCCCAGGC (SEQ 21 downstream ID NO: 1520)

Table 9D provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the fourth tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation andPAM is NNGRRT. It is contemplated herein that the targeting domainhybridizes to the target domain through complementary base pairing. Anyof the targeting domains in the table can be used with a S. aureus Cas9molecule that generates a double stranded break (Cas9 nuclease) or asingle-stranded break (Cas9 nickase).

TABLE 9D Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-710 − AAUGUAGAAUAAAUUUAUUUAAUG 24 upstream(SEQ ID NO: 1521) CEP290-711 − AUGUAGAAUAAAUUUAUUUAAUG 23 upstream (SEQID NO: 1522) CEP290-712 − AGAAUAAAUUUAUUUAAUG (SEQ 19 upstream ID NO:1523) CEP290-713 + AUUUAUUCUACAAUAAAAAAUGAU 24 upstream (SEQ ID NO:1524) CEP290-714 + AUUCUACAAUAAAAAAUGAU (SEQ 20 upstream ID NO: 1525)CEP290-715 − AGAGAAAAAGGAGCAUGAAACAGG 24 upstream (SEQ ID NO: 1526)CEP290-716 − AGAAAAAGGAGCAUGAAACAGG 22 upstream (SEQ ID NO: 1527)CEP290-717 − AAAAAGGAGCAUGAAACAGG (SEQ 20 upstream ID NO: 1528)CEP290-718 − AAAAGGAGCAUGAAACAGG (SEQ 19 upstream ID NO: 1529)CEP290-719 − AAAGGAGCAUGAAACAGG (SEQ ID 18 upstream NO: 1530)CEP290-720 + ACAAUAAAAAAUGAUGAGAAUUUA 24 upstream (SEQ ID NO: 1531)CEP290-721 + AAUAAAAAAUGAUGAGAAUUUA 22 upstream (SEQ ID NO: 1532)CEP290-722 + AUAAAAAAUGAUGAGAAUUUA 21 upstream (SEQ ID NO: 1533)CEP290-723 + AAAAAAUGAUGAGAAUUUA (SEQ 19 upstream ID NO: 1534)CEP290-724 + AAAAAUGAUGAGAAUUUA (SEQ ID 18 upstream NO: 1535) CEP290-725− AUGUAGAAUAAAAAAUAAAAAAAC 24 upstream (SEQ ID NO: 1536) CEP290-726 −AGAAUAAAAAAUAAAAAAAC (SEQ 20 upstream ID NO: 1537) CEP290-727 −AAUAAAAAAUAAAAAAAC (SEQ ID 18 upstream NO: 1538) CEP290-728 −AAUAAAAAAUAAAAAAACUAGAG 23 upstream (SEQ ID NO: 1539) CEP290-729 −AUAAAAAAUAAAAAAACUAGAG 22 upstream (SEQ ID NO: 1540) CEP290-730 −AAAAAAUAAAAAAACUAGAG (SEQ 20 upstream ID NO: 1541) CEP290-731 −AAAAAUAAAAAAACUAGAG (SEQ 19 upstream ID NO: 1542) CEP290-732 −AAAAUAAAAAAACUAGAG (SEQ ID 18 upstream NO: 1543) CEP290-733 +CAAUAAAAAAUGAUGAGAAUUUA 23 upstream (SEQ ID NO: 1544) CEP290-734 −UGUAGAAUAAAUUUAUUUAAUG 22 upstream (SEQ ID NO: 1545) CEP290-735 −UAGAAUAAAUUUAUUUAAUG (SEQ 20 upstream ID NO: 1546) CEP290-736 +UUUAUUCUACAAUAAAAAAUGAU 23 upstream (SEQ ID NO: 1547) CEP290-737 +UUAUUCUACAAUAAAAAAUGAU 22 upstream (SEQ ID NO: 1548) CEP290-738 +UAUUCUACAAUAAAAAAUGAU 21 upstream (SEQ ID NO: 1549) CEP290-739 +UUCUACAAUAAAAAAUGAU (SEQ 19 upstream ID NO: 1550) CEP290-740 +UCUACAAUAAAAAAUGAU (SEQ ID 18 upstream NO: 1551) CEP290-741 +UAAAAAAUGAUGAGAAUUUA (SEQ 20 upstream ID NO: 1552) CEP290-742 −UGUAGAAUAAAAAAUAAAAAAAC 23 upstream (SEQ ID NO: 1553) CEP290-743 −UAGAAUAAAAAAUAAAAAAAC 21 upstream (SEQ ID NO: 1554) CEP290-744 −UAAAAAAUAAAAAAACUAGAG 21 upstream (SEQ ID NO: 1555) CEP290-745 −AAAAGAAAUAGAUGUAGAUUGAGG 24 downstream (SEQ ID NO: 1556) CEP290-746 −AAAGAAAUAGAUGUAGAUUGAGG 23 downstream (SEQ ID NO: 1557) CEP290-747 −AAGAAAUAGAUGUAGAUUGAGG 22 downstream (SEQ ID NO: 1558) CEP290-748 −AGAAAUAGAUGUAGAUUGAGG 21 downstream (SEQ ID NO: 1559) CEP290-749 −AAAUAGAUGUAGAUUGAGG (SEQ 19 downstream ID NO: 1560) CEP290-750 −AAUAGAUGUAGAUUGAGG (SEQ ID 18 downstream NO: 1561) CEP290-751 −AUAAUAAGGAAAUACAAAAACUGG 24 downstream (SEQ ID NO: 1562) CEP290-752 −AAUAAGGAAAUACAAAAACUGG 22 downstream (SEQ ID NO: 1563) CEP290-753 −AUAAGGAAAUACAAAAACUGG 21 downstream (SEQ ID NO: 1564) CEP290-754 −AAGGAAAUACAAAAACUGG (SEQ 19 downstream ID NO: 1565) CEP290-755 −AGGAAAUACAAAAACUGG (SEQ ID 18 downstream NO: 1566) CEP290-756 −AUAGAUAAUAAGGAAAUACAAAAA 24 downstream (SEQ ID NO: 1567) CEP290-757 −AGAUAAUAAGGAAAUACAAAAA 22 downstream (SEQ ID NO: 1568) CEP290-758 −AUAAUAAGGAAAUACAAAAA (SEQ 20 downstream ID NO: 1569) CEP290-759 −AAUAAGGAAAUACAAAAA (SEQ ID 18 downstream NO: 1570) CEP290-760 +AAAAAAAAAAACAACAAAAA (SEQ 20 downstream ID NO: 1571) CEP290-761 +AAAAAAAAAACAACAAAAA (SEQ 19 downstream ID NO: 1572) CEP290-762 +AAAAAAAAACAACAAAAA (SEQ ID 18 downstream NO: 1573) CEP290-763 −AGAGUCUCACUGUGUUGCCCAGGC 24 downstream (SEQ ID NO: 1574) CEP290-764 −AGUCUCACUGUGUUGCCCAGGC 22 downstream (SEQ ID NO: 1575) CEP290-765 +CAAAAAAAAAAACAACAAAAA 21 downstream (SEQ ID NO: 1576) CEP290-766 −CUCACUGUGUUGCCCAGGC (SEQ ID 19 downstream NO: 1577) CEP290-767 −UAAUAAGGAAAUACAAAAACUGG 23 downstream (SEQ ID NO: 1578) CEP290-768 −UAAGGAAAUACAAAAACUGG (SEQ 20 downstream ID NO: 1579) CEP290-769 −UAGAUAAUAAGGAAAUACAAAAA 23 downstream (SEQ ID NO: 1580) CEP290-770 −UAAUAAGGAAAUACAAAAA (SEQ 19 downstream ID NO: 1581) CEP290-771 −UGUGUUGCCCAGGCUGGAGUGCAG 24 downstream (SEQ ID NO: 1582) CEP290-772 −UGUUGCCCAGGCUGGAGUGCAG 22 downstream (SEQ ID NO: 1583) CEP290-773 −UUGCCCAGGCUGGAGUGCAG (SEQ 20 downstream ID NO: 1189) CEP290-774 −UGCCCAGGCUGGAGUGCAG (SEQ ID 19 downstream NO: 1585) CEP290-775 +UUUCAAAAAAAAAAACAACAAAAA 24 downstream (SEQ ID NO: 1586) CEP290-776 +UUCAAAAAAAAAAACAACAAAAA 23 downstream (SEQ ID NO: 1587) CEP290-777 +UCAAAAAAAAAAACAACAAAAA 22 downstream (SEQ ID NO: 1588) CEP290-778 −UUUUUGUUGUUUUUUUUUUUGAAA 24 downstream (SEQ ID NO: 1589) CEP290-779 −UUUUGUUGUUUUUUUUUUUGAAA 23 downstream (SEQ ID NO: 1590) CEP290-780 −UUUGUUGUUUUUUUUUUUGAAA 22 downstream (SEQ ID NO: 1591) CEP290-781 −UUGUUGUUUUUUUUUUUGAAA 21 downstream (SEQ ID NO: 1592) CEP290-782 −UGUUGUUUUUUUUUUUGAAA (SEQ 20 downstream ID NO: 1593) CEP290-783 −UUGUUUUUUUUUUUGAAA (SEQ ID 18 downstream NO: 1594) CEP290-784 −UCUCACUGUGUUGCCCAGGC (SEQ 20 downstream ID NO: 1182) CEP290-785 −UCACUGUGUUGCCCAGGC (SEQ ID 18 downstream NO: 1596)

Table 9E provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the fifth tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation andPAM is NNGRRV. It is contemplated herein that the targeting domainhybridizes to the target domain through complementary base pairing. Anyof the targeting domains in the table can be used with a S. aureus Cas9molecule that generates a double stranded break (Cas9 nuclease) or asingle-stranded break (Cas9 nickase).

TABLE 9E Target Position DNA Site relative to gRNA Name Strand TargetingDomain Length mutation CEP290-786 + ACUGUUGGCUACAUCCAUUCC (SEQ ID 21upstream NO: 1597) CEP290-787 + AAUUUACAGAGUGCAUCCAUGGUC 24 upstream(SEQ ID NO: 1598) CEP290-788 + AUUUACAGAGUGCAUCCAUGGUC (SEQ 23 upstreamID NO: 1599) CEP290-789 + ACAGAGUGCAUCCAUGGUC (SEQ ID 19 upstream NO:1600) CEP290-790 − AGCAUUAGAAAGUCCUAGGC (SEQ ID 20 upstream NO: 823)CEP290-791 − AUGGUCCCUGGCUUUUGUUCC (SEQ ID 21 upstream NO: 1602)CEP290-792 − AUAGAGACACAUUCAGUAA (SEQ ID 19 upstream NO: 1603)CEP290-793 − AGCUCAAAAGCUUUUGCUGGCUCA 24 upstream (SEQ ID NO: 1604)CEP290-794 − AAAAGCUUUUGCUGGCUCA (SEQ ID 19 upstream NO: 1605)CEP290-795 − AAAGCUUUUGCUGGCUCA (SEQ ID NO: 18 upstream 1606)CEP290-796 + AAUCCAUAAGCCUCUAUUUCUGAU 24 upstream (SEQ ID NO: 1607)CEP290-797 + AUCCAUAAGCCUCUAUUUCUGAU (SEQ 23 upstream ID NO: 1608)CEP290-798 + AUAAGCCUCUAUUUCUGAU (SEQ ID 19 upstream NO: 1609)CEP290-799 + AGCUAAAUCAUGCAAGUGACCUA (SEQ 23 upstream ID NO: 1610)CEP290-800 + AAAUCAUGCAAGUGACCUA (SEQ ID 19 upstream NO: 1611)CEP290-801 + AAUCAUGCAAGUGACCUA (SEQ ID NO: 18 upstream 1612) CEP290-802− AAACCUCUUUUAACCAGACAUCU (SEQ 23 upstream ID NO: 1613) CEP290-803 −AACCUCUUUUAACCAGACAUCU (SEQ 22 upstream ID NO: 1614) CEP290-804 −ACCUCUUUUAACCAGACAUCU (SEQ ID 21 upstream NO: 1615) CEP290-805 +AGUUUGUUCUGGGUACAGGGGUAA 24 upstream (SEQ ID NO: 1616) CEP290-806 +AUGACUCAUAAUUUAGUAGGAAUC 24 upstream (SEQ ID NO: 1617) CEP290-807 +ACUCAUAAUUUAGUAGGAAUC (SEQ ID 21 upstream NO: 1618) CEP290-808 −AAUGGAUGUAGCCAACAGUAG (SEQ ID 21 upstream NO: 1619) CEP290-809 −AUGGAUGUAGCCAACAGUAG (SEQ ID 20 upstream NO: 1620) CEP290-810 +AUCACCUCUCUUUGGCAAAAGCAG 24 upstream (SEQ ID NO: 1621) CEP290-811 +ACCUCUCUUUGGCAAAAGCAG (SEQ ID 21 upstream NO: 1622) CEP290-812 −AGGUAGAAUAUUGUAAUCAAAGG 23 upstream (SEQ ID NO: 1623) CEP290-813 −AGAAUAUUGUAAUCAAAGG (SEQ ID 19 upstream NO: 1624) CEP290-814 +AAGGAACAAAAGCCAGGGACC (SEQ ID 21 upstream NO: 1625) CEP290-815 +AGGAACAAAAGCCAGGGACC (SEQ ID 20 upstream NO: 1626) CEP290-816 +ACAUCCAUUCCAAGGAACAAAAGC 24 upstream (SEQ ID NO: 1627) CEP290-817 +AUCCAUUCCAAGGAACAAAAGC (SEQ 22 upstream ID NO: 1628) CEP290-818 +AUUCCAAGGAACAAAAGC (SEQ ID NO: 18 upstream 1629) CEP290-819 +AGAAUUAGAUCUUAUUCUACUCCU 24 upstream (SEQ ID NO: 1630) CEP290-820 +AAUUAGAUCUUAUUCUACUCCU (SEQ 22 upstream ID NO: 1631) CEP290-821 +AUUAGAUCUUAUUCUACUCCU (SEQ ID 21 upstream NO: 1632) CEP290-822 +AGAUCUUAUUCUACUCCU (SEQ ID NO: 18 upstream 1633) CEP290-823 −AUUUGUUCAUCUUCCUCAU (SEQ ID 19 upstream NO: 1634) CEP290-824 −AGAGGUGAUUAUGUUACUUUUUA 23 upstream (SEQ ID NO: 1635) CEP290-825 −AGGUGAUUAUGUUACUUUUUA (SEQ 21 upstream ID NO: 1636) CEP290-826 −AACCUCUUUUAACCAGACAUCUAA 24 upstream (SEQ ID NO: 1637) CEP290-827 −ACCUCUUUUAACCAGACAUCUAA (SEQ 23 upstream ID NO: 1638) CEP290-828 +AUAAACAUGACUCAUAAUUUAG (SEQ 22 upstream ID NO: 1639) CEP290-829 +AAACAUGACUCAUAAUUUAG (SEQ ID 20 upstream NO: 805) CEP290-830 +AACAUGACUCAUAAUUUAG (SEQ ID 19 upstream NO: 1641) CEP290-831 +ACAUGACUCAUAAUUUAG (SEQ ID NO: 18 upstream 1642) CEP290-832 −ACAGGUAGAAUAUUGUAAUCAAAG 24 upstream (SEQ ID NO: 1643) CEP290-833 −AGGUAGAAUAUUGUAAUCAAAG (SEQ 22 upstream ID NO: 1644) CEP290-834 −AGAAUAUUGUAAUCAAAG (SEQ ID NO: 18 upstream 1645) CEP290-835 +AUAGUUUGUUCUGGGUACAGGGGU 24 upstream (SEQ ID NO: 1646) CEP290-836 +AGUUUGUUCUGGGUACAGGGGU (SEQ 22 upstream ID NO: 1647) CEP290-837 −AGACAUCUAAGAGAAAAAGGAGC 23 upstream (SEQ ID NO: 1648) CEP290-838 −ACAUCUAAGAGAAAAAGGAGC (SEQ ID 21 upstream NO: 1649) CEP290-839 −AUCUAAGAGAAAAAGGAGC (SEQ ID 19 upstream NO: 1650) CEP290-840 +AGAGGAUAGGACAGAGGACA (SEQ ID 20 upstream NO: 735) CEP290-841 +AGGAUAGGACAGAGGACA (SEQ ID NO: 18 upstream 1652) CEP290-842 +AGGAAAGAUGAAAAAUACUCUU (SEQ 22 upstream ID NO: 1653) CEP290-843 +AAAGAUGAAAAAUACUCUU (SEQ ID 19 upstream NO: 1654) CEP290-844 +AAGAUGAAAAAUACUCUU (SEQ ID NO: 18 upstream 1655) CEP290-845 +AGGAAAGAUGAAAAAUACUCUUU 23 upstream (SEQ ID NO: 1656) CEP290-846 +AAAGAUGAAAAAUACUCUUU (SEQ ID 20 upstream NO: 737) CEP290-847 +AAGAUGAAAAAUACUCUUU (SEQ ID 19 upstream NO: 1658) CEP290-848 +AGAUGAAAAAUACUCUUU (SEQ ID NO: 18 upstream 1659) CEP290-849 +AGGAAAGAUGAAAAAUACUCU (SEQ ID 21 upstream NO: 1660) CEP290-850 +AAAGAUGAAAAAUACUCU (SEQ ID NO: 18 upstream 1661) CEP290-851 +AUAGGACAGAGGACAUGGAGAA (SEQ 22 upstream ID NO: 1662) CEP290-852 +AGGACAGAGGACAUGGAGAA (SEQ ID 20 upstream NO: 736) CEP290-853 +AGGAUAGGACAGAGGACAUGGAGA 24 upstream (SEQ ID NO: 1664) CEP290-854 +AUAGGACAGAGGACAUGGAGA (SEQ ID 21 upstream NO: 1665) CEP290-855 +AGGACAGAGGACAUGGAGA (SEQ ID 19 upstream NO: 1666) CEP290-856 +AAGGAACAAAAGCCAGGGACCAU (SEQ 23 upstream ID NO: 1667) CEP290-857 +AGGAACAAAAGCCAGGGACCAU (SEQ 22 upstream ID NO: 1668) CEP290-858 +AACAAAAGCCAGGGACCAU (SEQ ID 19 upstream NO: 1669) CEP290-859 +ACAAAAGCCAGGGACCAU (SEQ ID NO: 18 upstream 1670) CEP290-860 +ACAUUUAUUCUACAAUAAAAAAUG 24 upstream (SEQ ID NO: 1671) CEP290-861 +AUUUAUUCUACAAUAAAAAAUG (SEQ 22 upstream ID NO: 1672) CEP290-862 +AUUCUACAAUAAAAAAUG (SEQ ID NO: 18 upstream 1673) CEP290-863 +AUUGUGUGUGUGUGUGUGUGUUAU 24 upstream (SEQ ID NO: 1674) CEP290-864 +CUACUGUUGGCUACAUCCAUUCC (SEQ 23 upstream ID NO: 1675) CEP290-865 +CUGUUGGCUACAUCCAUUCC (SEQ ID 20 upstream NO: 1676) CEP290-866 +CAGAGUGCAUCCAUGGUC (SEQ ID NO: 18 upstream 1677) CEP290-867 −CUCCAGCAUUAGAAAGUCCUAGGC 24 upstream (SEQ ID NO: 1678) CEP290-868 −CCAGCAUUAGAAAGUCCUAGGC (SEQ 22 upstream ID NO: 1679) CEP290-869 −CAGCAUUAGAAAGUCCUAGGC (SEQ ID 21 upstream NO: 1680) CEP290-870 −CAUUAGAAAGUCCUAGGC (SEQ ID NO: 18 upstream 1681) CEP290-871 −CCCAUGGUCCCUGGCUUUUGUUCC 24 upstream (SEQ ID NO: 1682) CEP290-872 −CCAUGGUCCCUGGCUUUUGUUCC (SEQ 23 upstream ID NO: 1683) CEP290-873 −CAUGGUCCCUGGCUUUUGUUCC (SEQ 22 upstream ID NO: 1684) CEP290-874 −CUCAUAGAGACACAUUCAGUAA (SEQ 22 upstream ID NO: 1685) CEP290-875 −CAUAGAGACACAUUCAGUAA (SEQ ID 20 upstream NO: 750) CEP290-876 −CUCAAAAGCUUUUGCUGGCUCA (SEQ 22 upstream ID NO: 1687) CEP290-877 −CAAAAGCUUUUGCUGGCUCA (SEQ ID 20 upstream NO: 762) CEP290-878 +CCAUAAGCCUCUAUUUCUGAU (SEQ ID 21 upstream NO: 1689) CEP290-879 +CAUAAGCCUCUAUUUCUGAU (SEQ ID 20 upstream NO: 851) CEP290-880 +CAGCUAAAUCAUGCAAGUGACCUA 24 upstream (SEQ ID NO: 1691) CEP290-881 +CUAAAUCAUGCAAGUGACCUA (SEQ ID 21 upstream NO: 1692) CEP290-882 −CAAACCUCUUUUAACCAGACAUCU 24 upstream (SEQ ID NO: 1693) CEP290-883 −CCUCUUUUAACCAGACAUCU (SEQ ID 20 upstream NO: 1694) CEP290-884 −CUCUUUUAACCAGACAUCU (SEQ ID 19 upstream NO: 1695) CEP290-885 +CUCAUAAUUUAGUAGGAAUC (SEQ ID 20 upstream NO: 864) CEP290-886 +CAUAAUUUAGUAGGAAUC (SEQ ID NO: 18 upstream 1697) CEP290-887 +CACCUCUCUUUGGCAAAAGCAG (SEQ 22 upstream ID NO: 1698) CEP290-888 +CCUCUCUUUGGCAAAAGCAG (SEQ ID 20 upstream NO: 859) CEP290-889 +CUCUCUUUGGCAAAAGCAG (SEQ ID 19 upstream NO: 1700) CEP290-890 −CAGGUAGAAUAUUGUAAUCAAAGG 24 upstream (SEQ ID NO: 1701) CEP290-891 +CCAAGGAACAAAAGCCAGGGACC (SEQ 23 upstream ID NO: 1702) CEP290-892 +CAAGGAACAAAAGCCAGGGACC (SEQ 22 upstream ID NO: 1703) CEP290-893 +CAUCCAUUCCAAGGAACAAAAGC (SEQ 23 upstream ID NO: 1704) CEP290-894 +CCAUUCCAAGGAACAAAAGC (SEQ ID 20 upstream NO: 1705) CEP290-895 +CAUUCCAAGGAACAAAAGC (SEQ ID 19 upstream NO: 1706) CEP290-896 +CUCUUGCCUAGGACUUUCUAAUGC 24 upstream (SEQ ID NO: 1707) CEP290-897 +CUUGCCUAGGACUUUCUAAUGC (SEQ 22 upstream ID NO: 1708) CEP290-898 +CCUAGGACUUUCUAAUGC (SEQ ID NO: 18 upstream 1709) CEP290-899 −CCUGAUUUGUUCAUCUUCCUCAU (SEQ 23 upstream ID NO: 1710) CEP290-900 −CUGAUUUGUUCAUCUUCCUCAU (SEQ 22 upstream ID NO: 1711) CEP290-901 −CCUCUUUUAACCAGACAUCUAA (SEQ 22 upstream ID NO: 1712) CEP290-902 −CUCUUUUAACCAGACAUCUAA (SEQ ID 21 upstream NO: 1713) CEP290-903 −CUUUUAACCAGACAUCUAA (SEQ ID 19 upstream NO: 1714) CEP290-904 −CCUCUGUCCUAUCCUCUCCAGCAU 24 upstream (SEQ ID NO: 1715) CEP290-905 −CUCUGUCCUAUCCUCUCCAGCAU (SEQ 23 upstream ID NO: 1716) CEP290-906 −CUGUCCUAUCCUCUCCAGCAU (SEQ ID 21 upstream NO: 1717) CEP290-907 −CAGGUAGAAUAUUGUAAUCAAAG 23 upstream (SEQ ID NO: 1718) CEP290-908 +CUGGGUACAGGGGUAAGAGA (SEQ ID 20 upstream NO: 1719) CEP290-909 −CUUUCUGCUGCUUUUGCCA (SEQ ID 19 upstream NO: 1720) CEP290-910 −CAGACAUCUAAGAGAAAAAGGAGC 24 upstream (SEQ ID NO: 1721) CEP290-911 −CAUCUAAGAGAAAAAGGAGC (SEQ ID 20 upstream NO: 1722) CEP290-912 +CUGGAGAGGAUAGGACAGAGGACA 24 upstream (SEQ ID NO: 1723) CEP290-913 +CAAGGAACAAAAGCCAGGGACCAU 24 upstream (SEQ ID NO: 1724) CEP290-914 +CAUUUAUUCUACAAUAAAAAAUG 23 upstream (SEQ ID NO: 1725) CEP290-915 +GCUACUGUUGGCUACAUCCAUUCC 24 upstream (SEQ ID NO: 1726) CEP290-916 +GUUGGCUACAUCCAUUCC (SEQ ID NO: 18 upstream 1727) CEP290-917 −GCAUUAGAAAGUCCUAGGC (SEQ ID 19 upstream NO: 1728) CEP290-918 −GGUCCCUGGCUUUUGUUCC (SEQ ID 19 upstream NO: 1729) CEP290-919 −GUCCCUGGCUUUUGUUCC (SEQ ID NO: 18 upstream 1730) CEP290-920 −GGCUCAUAGAGACACAUUCAGUAA 24 upstream (SEQ ID NO: 1731) CEP290-921 −GCUCAUAGAGACACAUUCAGUAA (SEQ 23 upstream ID NO: 1732) CEP290-922 −GCUCAAAAGCUUUUGCUGGCUCA (SEQ 23 upstream ID NO: 1733) CEP290-923 +GCUAAAUCAUGCAAGUGACCUA (SEQ 22 upstream ID NO: 1734) CEP290-924 +GUUUGUUCUGGGUACAGGGGUAA 23 upstream (SEQ ID NO: 1735) CEP290-925 +GUUCUGGGUACAGGGGUAA (SEQ ID 19 upstream NO: 1736) CEP290-926 +GACUCAUAAUUUAGUAGGAAUC (SEQ 22 upstream ID NO: 1737) CEP290-927 −GGAAUGGAUGUAGCCAACAGUAG 23 upstream (SEQ ID NO: 1738) CEP290-928 −GAAUGGAUGUAGCCAACAGUAG (SEQ 22 upstream ID NO: 1739) CEP290-929 −GGAUGUAGCCAACAGUAG (SEQ ID NO: 18 upstream 1740) CEP290-930 −GGUAGAAUAUUGUAAUCAAAGG (SEQ 22 upstream ID NO: 1741) CEP290-931 −GUAGAAUAUUGUAAUCAAAGG (SEQ 21 upstream ID NO: 1742) CEP290-932 −GAAUAUUGUAAUCAAAGG (SEQ ID NO: 18 upstream 1743) CEP290-933 +GGAACAAAAGCCAGGGACC (SEQ ID 19 upstream NO: 1744) CEP290-934 +GAACAAAAGCCAGGGACC (SEQ ID NO: 18 upstream 1745) CEP290-935 +GAAUUAGAUCUUAUUCUACUCCU (SEQ 23 upstream ID NO: 1746) CEP290-936 +GCCUAGGACUUUCUAAUGC (SEQ ID 19 upstream NO: 1747) CEP290-937 −GAUUUGUUCAUCUUCCUCAU (SEQ ID 20 upstream NO: 774) CEP290-938 −GAGAGGUGAUUAUGUUACUUUUUA 24 upstream (SEQ ID NO: 1749) CEP290-939 −GAGGUGAUUAUGUUACUUUUUA (SEQ 22 upstream ID NO: 1750) CEP290-940 −GGUGAUUAUGUUACUUUUUA (SEQ ID 20 upstream NO: 780) CEP290-941 −GUGAUUAUGUUACUUUUUA (SEQ ID 19 upstream NO: 1752) CEP290-942 −GUCCUAUCCUCUCCAGCAU (SEQ ID 19 upstream NO: 1753) CEP290-943 +GAUAAACAUGACUCAUAAUUUAG 23 upstream (SEQ ID NO: 1754) CEP290-944 −GGUAGAAUAUUGUAAUCAAAG (SEQ 21 upstream ID NO: 1755) CEP290-945 −GUAGAAUAUUGUAAUCAAAG (SEQ ID 20 upstream NO: 1756) CEP290-946 +GUUCUGGGUACAGGGGUAAGAGA 23 upstream (SEQ ID NO: 1757) CEP290-947 +GGGUACAGGGGUAAGAGA (SEQ ID NO: 18 upstream 1758) CEP290-948 +GUUUGUUCUGGGUACAGGGGU (SEQ ID 21 upstream NO: 1759) CEP290-949 −GUUUGCUUUCUGCUGCUUUUGCCA 24 upstream (SEQ ID NO: 1760) CEP290-950 −GCUUUCUGCUGCUUUUGCCA (SEQ ID 20 upstream NO: 776) CEP290-951 −GACAUCUAAGAGAAAAAGGAGC (SEQ 22 upstream ID NO: 1762) CEP290-952 +GGAGAGGAUAGGACAGAGGACA (SEQ 22 upstream ID NO: 1763) CEP290-953 +GAGAGGAUAGGACAGAGGACA (SEQ ID 21 upstream NO: 1764) CEP290-954 +GAGGAUAGGACAGAGGACA (SEQ ID 19 upstream NO: 1765) CEP290-955 +GGAAAGAUGAAAAAUACUCUU (SEQ ID 21 upstream NO: 1766) CEP290-956 +GAAAGAUGAAAAAUACUCUU (SEQ ID 20 upstream NO: 462) CEP290-957 +GGAAAGAUGAAAAAUACUCUUU (SEQ 22 upstream ID NO: 1767) CEP290-958 +GAAAGAUGAAAAAUACUCUUU (SEQ ID 21 upstream NO: 1768) CEP290-959 +GGAAAGAUGAAAAAUACUCU (SEQ ID 20 upstream NO: 778) CEP290-960 +GAAAGAUGAAAAAUACUCU (SEQ ID 19 upstream NO: 1770) CEP290-961 +GGAUAGGACAGAGGACAUGGAGAA 24 upstream (SEQ ID NO: 1771) CEP290-962 +GAUAGGACAGAGGACAUGGAGAA 23 upstream (SEQ ID NO: 1772) CEP290-963 +GGACAGAGGACAUGGAGAA (SEQ ID 19 upstream NO: 1773) CEP290-964 +GACAGAGGACAUGGAGAA (SEQ ID NO: 18 upstream 1774) CEP290-965 +GGAUAGGACAGAGGACAUGGAGA 23 upstream (SEQ ID NO: 1775) CEP290-966 +GAUAGGACAGAGGACAUGGAGA (SEQ 22 upstream ID NO: 1776) CEP290-967 +GGACAGAGGACAUGGAGA (SEQ ID NO: 18 upstream 1777) CEP290-968 +GGAACAAAAGCCAGGGACCAU (SEQ ID 21 upstream NO: 1778) CEP290-969 +GAACAAAAGCCAGGGACCAU (SEQ ID 20 upstream NO: 465) CEP290-970 +GUGUGUGUGUGUGUGUGUUAU (SEQ 21 upstream ID NO: 1779) CEP290-971 +GUGUGUGUGUGUGUGUUAU (SEQ ID 19 upstream NO: 1780) CEP290-972 +GUGUGUGUGUGUGUGUGUUAUG (SEQ 22 upstream ID NO: 1781) CEP290-973 +GUGUGUGUGUGUGUGUUAUG (SEQ ID 20 upstream NO: 1154) CEP290-974 +GUGUGUGUGUGUGUUAUG (SEQ ID NO: 18 upstream 1783) CEP290-975 +UACUGUUGGCUACAUCCAUUCC (SEQ 22 upstream ID NO: 1784) CEP290-976 +UGUUGGCUACAUCCAUUCC (SEQ ID 19 upstream NO: 1785) CEP290-977 +UUUACAGAGUGCAUCCAUGGUC (SEQ 22 upstream ID NO: 1786) CEP290-978 +UUACAGAGUGCAUCCAUGGUC (SEQ ID 21 upstream NO: 1787) CEP290-979 +UACAGAGUGCAUCCAUGGUC (SEQ ID 20 upstream NO: 1788) CEP290-980 −UCCAGCAUUAGAAAGUCCUAGGC (SEQ 23 upstream ID NO: 1789) CEP290-981 −UGGUCCCUGGCUUUUGUUCC (SEQ ID 20 upstream NO: 1790) CEP290-982 −UCAUAGAGACACAUUCAGUAA (SEQ ID 21 upstream NO: 1791) CEP290-983 −UAGAGACACAUUCAGUAA (SEQ ID NO: 18 upstream 1792) CEP290-984 −UCAAAAGCUUUUGCUGGCUCA (SEQ ID 21 upstream NO: 1793) CEP290-985 +UCCAUAAGCCUCUAUUUCUGAU (SEQ 22 upstream ID NO: 1794) CEP290-986 +UAAGCCUCUAUUUCUGAU (SEQ ID NO: 18 upstream 1795) CEP290-987 +UAAAUCAUGCAAGUGACCUA (SEQ ID 20 upstream NO: 508) CEP290-988 −UCUUUUAACCAGACAUCU (SEQ ID NO: 18 upstream 1796) CEP290-989 +UUUGUUCUGGGUACAGGGGUAA (SEQ 22 upstream ID NO: 1797) CEP290-990 +UUGUUCUGGGUACAGGGGUAA (SEQ ID 21 upstream NO: 1798) CEP290-991 +UGUUCUGGGUACAGGGGUAA (SEQ ID 20 upstream NO: 1799) CEP290-992 +UUCUGGGUACAGGGGUAA (SEQ ID NO: 18 upstream 1800) CEP290-993 +UGACUCAUAAUUUAGUAGGAAUC 23 upstream (SEQ ID NO: 1801) CEP290-994 +UCAUAAUUUAGUAGGAAUC (SEQ ID 19 upstream NO: 1802) CEP290-995 −UGGAAUGGAUGUAGCCAACAGUAG 24 upstream (SEQ ID NO: 1803) CEP290-996 −UGGAUGUAGCCAACAGUAG (SEQ ID 19 upstream NO: 1804) CEP290-997 +UCACCUCUCUUUGGCAAAAGCAG (SEQ 23 upstream ID NO: 1805) CEP290-998 +UCUCUUUGGCAAAAGCAG (SEQ ID NO: 18 upstream 1806) CEP290-999 −UAGAAUAUUGUAAUCAAAGG (SEQ ID 20 upstream NO: 1807) CEP290- +UCCAAGGAACAAAAGCCAGGGACC 24 upstream 1000 (SEQ ID NO: 1808) CEP290- +UCCAUUCCAAGGAACAAAAGC (SEQ ID 21 upstream 1001 NO: 1809) CEP290- +UUAGAUCUUAUUCUACUCCU (SEQ ID 20 upstream 1002 NO: 902) CEP290- +UAGAUCUUAUUCUACUCCU (SEQ ID 19 upstream 1003 NO: 1811) CEP290- +UCUUGCCUAGGACUUUCUAAUGC (SEQ 23 upstream 1004 ID NO: 1812) CEP290- +UUGCCUAGGACUUUCUAAUGC (SEQ ID 21 upstream 1005 NO: 1813) CEP290- +UGCCUAGGACUUUCUAAUGC (SEQ ID 20 upstream 1006 NO: 632) CEP290- −UCCUGAUUUGUUCAUCUUCCUCAU 24 upstream 1007 (SEQ ID NO: 1814) CEP290- −UGAUUUGUUCAUCUUCCUCAU (SEQ ID 21 upstream 1008 NO: 1815) CEP290- −UUUGUUCAUCUUCCUCAU (SEQ ID NO: 18 upstream 1009 1816) CEP290- −UGAUUAUGUUACUUUUUA (SEQ ID NO: 18 upstream 1010 1817) CEP290- −UCUUUUAACCAGACAUCUAA (SEQ ID 20 upstream 1011 NO: 1818) CEP290- −UUUUAACCAGACAUCUAA (SEQ ID NO: 18 upstream 1012 1819) CEP290- −UCUGUCCUAUCCUCUCCAGCAU (SEQ 22 upstream 1013 ID NO: 1820) CEP290- −UGUCCUAUCCUCUCCAGCAU (SEQ ID 20 upstream 1014 NO: 899) CEP290- −UCCUAUCCUCUCCAGCAU (SEQ ID NO: 18 upstream 1015 1822) CEP290- +UGAUAAACAUGACUCAUAAUUUAG 24 upstream 1016 (SEQ ID NO: 1823) CEP290- +UAAACAUGACUCAUAAUUUAG (SEQ ID 21 upstream 1017 NO: 1824) CEP290- −UAGAAUAUUGUAAUCAAAG (SEQ ID 19 upstream 1018 NO: 1825) CEP290- +UGUUCUGGGUACAGGGGUAAGAGA 24 upstream 1019 (SEQ ID NO: 1826) CEP290- +UUCUGGGUACAGGGGUAAGAGA (SEQ 22 upstream 1020 ID NO: 1827) CEP290- +UCUGGGUACAGGGGUAAGAGA (SEQ ID 21 upstream 1021 NO: 1828) CEP290- +UGGGUACAGGGGUAAGAGA (SEQ ID 19 upstream 1022 NO: 1829) CEP290- +UAGUUUGUUCUGGGUACAGGGGU 23 upstream 1023 (SEQ ID NO: 1830) CEP290- +UUUGUUCUGGGUACAGGGGU (SEQ ID 20 upstream 1024 NO: 1831) CEP290- +UUGUUCUGGGUACAGGGGU (SEQ ID 19 upstream 1025 NO: 1832) CEP290- +UGUUCUGGGUACAGGGGU (SEQ ID NO: 18 upstream 1026 1833) CEP290- −UUUGCUUUCUGCUGCUUUUGCCA (SEQ 23 upstream 1027 ID NO: 1834) CEP290- −UUGCUUUCUGCUGCUUUUGCCA (SEQ 22 upstream 1028 ID NO: 1835) CEP290- −UGCUUUCUGCUGCUUUUGCCA (SEQ ID 21 upstream 1029 NO: 1836) CEP290- −UUUCUGCUGCUUUUGCCA (SEQ ID NO: 18 upstream 1030 1837) CEP290- −UCUAAGAGAAAAAGGAGC (SEQ ID NO: 18 upstream 1031 1838) CEP290- +UGGAGAGGAUAGGACAGAGGACA 23 upstream 1032 (SEQ ID NO: 1839) CEP290- +UUAGGAAAGAUGAAAAAUACUCUU 24 upstream 1033 (SEQ ID NO: 1840) CEP290- +UAGGAAAGAUGAAAAAUACUCUU 23 upstream 1034 (SEQ ID NO: 1841) CEP290- +UAGGAAAGAUGAAAAAUACUCUUU 24 upstream 1035 (SEQ ID NO: 1842) CEP290- +UUUAGGAAAGAUGAAAAAUACUCU 24 upstream 1036 (SEQ ID NO: 1843) CEP290- +UUAGGAAAGAUGAAAAAUACUCU 23 upstream 1037 (SEQ ID NO: 1844) CEP290- +UAGGAAAGAUGAAAAAUACUCU (SEQ 22 upstream 1038 ID NO: 1845) CEP290- +UAGGACAGAGGACAUGGAGAA (SEQ ID 21 upstream 1039 NO: 1846) CEP290- +UAGGACAGAGGACAUGGAGA (SEQ ID 20 upstream 1040 NO: 881) CEP290- +UUUAUUCUACAAUAAAAAAUG (SEQ ID 21 upstream 1041 NO: 1848) CEP290- +UUAUUCUACAAUAAAAAAUG (SEQ ID 20 upstream 1042 NO: 1849) CEP290- +UAUUCUACAAUAAAAAAUG (SEQ ID 19 upstream 1043 NO: 1850) CEP290- +UUGUGUGUGUGUGUGUGUGUUAU 23 upstream 1044 (SEQ ID NO: 1851) CEP290- +UGUGUGUGUGUGUGUGUGUUAU (SEQ 22 upstream 1045 ID NO: 1852) CEP290- +UGUGUGUGUGUGUGUGUUAU (SEQ ID 20 upstream 1046 NO: 1853) CEP290- +UGUGUGUGUGUGUGUUAU (SEQ ID NO: 18 upstream 1047 1854) CEP290- +UUGUGUGUGUGUGUGUGUGUUAUG 24 upstream 1048 (SEQ ID NO: 1855) CEP290- +UGUGUGUGUGUGUGUGUGUUAUG 23 upstream 1049 (SEQ ID NO: 1856) CEP290- +UGUGUGUGUGUGUGUGUUAUG (SEQ 21 upstream 1050 ID NO: 1857) CEP290- +UGUGUGUGUGUGUGUUAUG (SEQ ID 19 upstream 1051 NO: 1858) CEP290- +ACUGUUGGCUACAUCCAUUCCA (SEQ 22 upstream 1052 ID NO: 1859) CEP290- +AUUAUCCACAAGAUGUCUCUUGCC 24 upstream 1053 (SEQ ID NO: 1860) CEP290- +AUCCACAAGAUGUCUCUUGCC (SEQ ID 21 upstream 1054 NO: 1861) CEP290- +AUGAGCCAGCAAAAGCUU (SEQ ID NO: 18 upstream 1055 1862) CEP290- +ACAGAGUGCAUCCAUGGUCCAGG (SEQ 23 upstream 1056 ID NO: 1863) CEP290- +AGAGUGCAUCCAUGGUCCAGG (SEQ ID 21 upstream 1057 NO: 1864) CEP290- +AGUGCAUCCAUGGUCCAGG (SEQ ID 19 upstream 1058 NO: 1865) CEP290- −AGCUGAAAUAUUAAGGGCUCUUC (SEQ 23 upstream 1059 ID NO: 1866) CEP290- −AAAUAUUAAGGGCUCUUC (SEQ ID NO: 18 upstream 1060 1867) CEP290- −AACUCUAUACCUUUUACUGAGGA (SEQ 23 upstream 1061 ID NO: 1868) CEP290- −ACUCUAUACCUUUUACUGAGGA (SEQ 22 upstream 1062 ID NO: 1869) CEP290- −ACUUGAACUCUAUACCUUUUACU (SEQ 23 upstream 1063 ID NO: 1870) CEP290- −AACUCUAUACCUUUUACU (SEQ ID NO: 18 upstream 1064 1871) CEP290- +AGUAGGAAUCCUGAAAGCUACU (SEQ 22 upstream 1065 ID NO: 1872) CEP290- +AGGAAUCCUGAAAGCUACU (SEQ ID 19 upstream 1066 NO: 1873) CEP290- −AGCCAACAGUAGCUGAAAUAUU (SEQ 22 upstream 1067 ID NO: 1874) CEP290- −AACAGUAGCUGAAAUAUU (SEQ ID NO: 18 upstream 1068 1875) CEP290- +AUCCAUUCCAAGGAACAAAAGCC (SEQ 23 upstream 1069 ID NO: 1876) CEP290- +AUUCCAAGGAACAAAAGCC (SEQ ID 19 upstream 1070 NO: 1877) CEP290- −AUCCCUUUCUCUUACCCCUGUACC 24 upstream 1071 (SEQ ID NO: 1878) CEP290- +AGGACUUUCUAAUGCUGGAGAGGA 24 upstream 1072 (SEQ ID NO: 1879) CEP290- +ACUUUCUAAUGCUGGAGAGGA (SEQ ID 21 upstream 1073 NO: 1880) CEP290- +AAUGCUGGAGAGGAUAGGACA (SEQ ID 21 upstream 1074 NO: 1881) CEP290- +AUGCUGGAGAGGAUAGGACA (SEQ ID 20 upstream 1075 NO: 838) CEP290- −AUCAUAAGUUACAAUCUGUGAAU 23 upstream 1076 (SEQ ID NO: 1883) CEP290- −AUAAGUUACAAUCUGUGAAU (SEQ ID 20 upstream 1077 NO: 1884) CEP290- −AAGUUACAAUCUGUGAAU (SEQ ID NO: 18 upstream 1078 1885) CEP290- −AACCAGACAUCUAAGAGAAAA (SEQ ID 21 upstream 1079 NO: 1886) CEP290- −ACCAGACAUCUAAGAGAAAA (SEQ ID 20 upstream 1080 NO: 1087) CEP290- +AAGCCUCUAUUUCUGAUGAGGAAG 24 upstream 1081 (SEQ ID NO: 1888) CEP290- +AGCCUCUAUUUCUGAUGAGGAAG (SEQ 23 upstream 1082 ID NO: 1889) CEP290- +AUGAGGAAGAUGAACAAAUC (SEQ ID 20 upstream 1083 NO: 733) CEP290- +AUUUACUGAAUGUGUCUCU (SEQ ID 19 upstream 1084 NO: 1891) CEP290- +ACAGGGGUAAGAGAAAGGG (SEQ ID 19 upstream 1085 NO: 1892) CEP290- +CUACUGUUGGCUACAUCCAUUCCA 24 upstream 1086 (SEQ ID NO: 1893) CEP290- +CUGUUGGCUACAUCCAUUCCA (SEQ ID 21 upstream 1087 NO: 1894) CEP290- +CCACAAGAUGUCUCUUGCC (SEQ ID 19 upstream 1088 NO: 1895) CEP290- +CACAAGAUGUCUCUUGCC (SEQ ID NO: 18 upstream 1089 1896) CEP290- −CCUUUGUAGUUAUCUUACAGCCAC 24 upstream 1090 (SEQ ID NO: 1897) CEP290- −CUUUGUAGUUAUCUUACAGCCAC (SEQ 23 upstream 1091 ID NO: 1898) CEP290- +CUCUAUGAGCCAGCAAAAGCUU (SEQ 22 upstream 1092 ID NO: 1899) CEP290- +CUAUGAGCCAGCAAAAGCUU (SEQ ID 20 upstream 1093 NO: 748) CEP290- +CAGAGUGCAUCCAUGGUCCAGG (SEQ 22 upstream 1094 ID NO: 1901) CEP290- −CUGAAAUAUUAAGGGCUCUUC (SEQ ID 21 upstream 1095 NO: 1902) CEP290- −CUCUAUACCUUUUACUGAGGA (SEQ ID 21 upstream 1096 NO: 1903) CEP290- −CUAUACCUUUUACUGAGGA (SEQ ID 19 upstream 1097 NO: 1904) CEP290- −CACUUGAACUCUAUACCUUUUACU 24 upstream 1098 (SEQ ID NO: 1905) CEP290- −CUUGAACUCUAUACCUUUUACU (SEQ 22 upstream 1099 ID NO: 1906) CEP290- −CCAACAGUAGCUGAAAUAUU (SEQ ID 20 upstream 1100 NO: 1907) CEP290- −CAACAGUAGCUGAAAUAUU (SEQ ID 19 upstream 1101 NO: 1908) CEP290- +CAUCCAUUCCAAGGAACAAAAGCC 24 upstream 1102 (SEQ ID NO: 1909) CEP290- +CCAUUCCAAGGAACAAAAGCC (SEQ ID 21 upstream 1103 NO: 1910) CEP290- +CAUUCCAAGGAACAAAAGCC (SEQ ID 20 upstream 1104 NO: 1131) CEP290- −CCCUUUCUCUUACCCCUGUACC (SEQ 22 upstream 1105 ID NO: 1912) CEP290- −CCUUUCUCUUACCCCUGUACC (SEQ ID 21 upstream 1106 NO: 1913) CEP290- −CUUUCUCUUACCCCUGUACC (SEQ ID 20 upstream 1107 NO: 1914) CEP290- +CUUUCUAAUGCUGGAGAGGA (SEQ ID 20 upstream 1108 NO: 869) CEP290- +CUAAUGCUGGAGAGGAUAGGACA 23 upstream 1109 (SEQ ID NO: 1916) CEP290- −CAUAAGUUACAAUCUGUGAAU (SEQ ID 21 upstream 1110 NO: 1917) CEP290- −CCAGACAUCUAAGAGAAAA (SEQ ID 19 upstream 1111 NO: 1918) CEP290- −CAGACAUCUAAGAGAAAA (SEQ ID NO: 18 upstream 1112 1919) CEP290- +CCUCUAUUUCUGAUGAGGAAG (SEQ ID 21 upstream 1113 NO: 1920) CEP290- +CUCUAUUUCUGAUGAGGAAG (SEQ ID 20 upstream 1114 NO: 866) CEP290- +CUAUUUCUGAUGAGGAAG (SEQ ID NO: 18 upstream 1115 1922) CEP290- +CUGAUGAGGAAGAUGAACAAAUC 23 upstream 1116 (SEQ ID NO: 1923) CEP290- +CAUUUACUGAAUGUGUCUCU (SEQ ID 20 upstream 1117 NO: 856) CEP290- +CAGGGGUAAGAGAAAGGG (SEQ ID NO: 18 upstream 1118 1925) CEP290- +GUUGGCUACAUCCAUUCCA (SEQ ID 19 upstream 1119 NO: 1926) CEP290- −GUAGUUAUCUUACAGCCAC (SEQ ID 19 upstream 1120 NO: 1927) CEP290- +GUCUCUAUGAGCCAGCAAAAGCUU 24 upstream 1121 (SEQ ID NO: 1928) CEP290- +GAGUGCAUCCAUGGUCCAGG (SEQ ID 20 upstream 1122 NO: 1929) CEP290- +GUGCAUCCAUGGUCCAGG (SEQ ID NO: 18 upstream 1123 1930) CEP290- −GCUGAAAUAUUAAGGGCUCUUC (SEQ 22 upstream 1124 ID NO: 1931) CEP290- −GAAAUAUUAAGGGCUCUUC (SEQ ID 19 upstream 1125 NO: 1932) CEP290- −GAACUCUAUACCUUUUACUGAGGA 24 upstream 1126 (SEQ ID NO: 1933) CEP290- −GAACUCUAUACCUUUUACU (SEQ ID 19 upstream 1127 NO: 1934) CEP290- +GUAGGAAUCCUGAAAGCUACU (SEQ ID 21 upstream 1128 NO: 1935) CEP290- +GGAAUCCUGAAAGCUACU (SEQ ID NO: 18 upstream 1129 1936) CEP290- −GUAGCCAACAGUAGCUGAAAUAUU 24 upstream 1130 (SEQ ID NO: 1937) CEP290- −GCCAACAGUAGCUGAAAUAUU (SEQ ID 21 upstream 1131 NO: 1938) CEP290- +GGACUUUCUAAUGCUGGAGAGGA 23 upstream 1132 (SEQ ID NO: 1939) CEP290- +GACUUUCUAAUGCUGGAGAGGA (SEQ 22 upstream 1133 ID NO: 1940) CEP290- +GCUGGAGAGGAUAGGACA (SEQ ID NO: 18 upstream 1134 1941) CEP290- +GCCUCUAUUUCUGAUGAGGAAG (SEQ 22 upstream 1135 ID NO: 1942) CEP290- +GAUGAGGAAGAUGAACAAAUC (SEQ ID 21 upstream 1136 NO: 1943) CEP290- +GAGGAAGAUGAACAAAUC (SEQ ID NO: 18 upstream 1137 1944) CEP290- +GGGUACAGGGGUAAGAGAAAGGG 23 upstream 1138 (SEQ ID NO: 1945) CEP290- +GGUACAGGGGUAAGAGAAAGGG (SEQ 22 upstream 1139 ID NO: 1946) CEP290- +GUACAGGGGUAAGAGAAAGGG (SEQ 21 upstream 1140 ID NO: 1947) CEP290- +GUGUGUGUGUGUGUGUGUUAUGU 23 upstream 1141 (SEQ ID NO: 1948) CEP290- +GUGUGUGUGUGUGUGUUAUGU (SEQ 21 upstream 1142 ID NO: 1949) CEP290- +GUGUGUGUGUGUGUUAUGU (SEQ ID 19 upstream 1143 NO: 1950) CEP290- +UACUGUUGGCUACAUCCAUUCCA (SEQ 23 upstream 1144 ID NO: 1951) CEP290- +UGUUGGCUACAUCCAUUCCA (SEQ ID 20 upstream 1145 NO: 1952) CEP290- +UUGGCUACAUCCAUUCCA (SEQ ID NO: 18 upstream 1146 1953) CEP290- +UUAUCCACAAGAUGUCUCUUGCC (SEQ 23 upstream 1147 ID NO: 1954) CEP290- +UAUCCACAAGAUGUCUCUUGCC (SEQ 22 upstream 1148 ID NO: 1955) CEP290- +UCCACAAGAUGUCUCUUGCC (SEQ ID 20 upstream 1149 NO: 885) CEP290- −UUUGUAGUUAUCUUACAGCCAC (SEQ 22 upstream 1150 ID NO: 1957) CEP290- −UUGUAGUUAUCUUACAGCCAC (SEQ ID 21 upstream 1151 NO: 1958) CEP290- −UGUAGUUAUCUUACAGCCAC (SEQ ID 20 upstream 1152 NO: 1959) CEP290- −UAGUUAUCUUACAGCCAC (SEQ ID NO: 18 upstream 1153 1960) CEP290- +UCUCUAUGAGCCAGCAAAAGCUU (SEQ 23 upstream 1154 ID NO: 1961) CEP290- +UCUAUGAGCCAGCAAAAGCUU (SEQ ID 21 upstream 1155 NO: 1962) CEP290- +UAUGAGCCAGCAAAAGCUU (SEQ ID 19 upstream 1156 NO: 1963) CEP290- +UACAGAGUGCAUCCAUGGUCCAGG 24 upstream 1157 (SEQ ID NO: 1964) CEP290- −UAGCUGAAAUAUUAAGGGCUCUUC 24 upstream 1158 (SEQ ID NO: 1965) CEP290- −UGAAAUAUUAAGGGCUCUUC (SEQ ID 20 upstream 1159 NO: 1966) CEP290- −UCUAUACCUUUUACUGAGGA (SEQ ID 20 upstream 1160 NO: 889) CEP290- −UAUACCUUUUACUGAGGA (SEQ ID NO: 18 upstream 1161 1968) CEP290- −UUGAACUCUAUACCUUUUACU (SEQ ID 21 upstream 1162 NO: 1969) CEP290- −UGAACUCUAUACCUUUUACU (SEQ ID 20 upstream 1163 NO: 1970) CEP290- +UUAGUAGGAAUCCUGAAAGCUACU 24 upstream 1164 (SEQ ID NO: 1971) CEP290- +UAGUAGGAAUCCUGAAAGCUACU (SEQ 23 upstream 1165 ID NO: 1972) CEP290- +UAGGAAUCCUGAAAGCUACU (SEQ ID 20 upstream 1166 NO: 760) CEP290- −UAGCCAACAGUAGCUGAAAUAUU (SEQ 23 upstream 1167 ID NO: 1974) CEP290- +UCCAUUCCAAGGAACAAAAGCC (SEQ 22 upstream 1168 ID NO: 1975) CEP290- +UUCCAAGGAACAAAAGCC (SEQ ID NO: 18 upstream 1169 1976) CEP290- −UCCCUUUCUCUUACCCCUGUACC (SEQ 23 upstream 1170 ID NO: 1977) CEP290- −UUUCUCUUACCCCUGUACC (SEQ ID 19 upstream 1171 NO: 1978) CEP290- −UUCUCUUACCCCUGUACC (SEQ ID NO: 18 upstream 1172 1979) CEP290- +UUUCUAAUGCUGGAGAGGA (SEQ ID 19 upstream 1173 NO: 1980) CEP290- +UUCUAAUGCUGGAGAGGA (SEQ ID NO: 18 upstream 1174 1981) CEP290- +UCUAAUGCUGGAGAGGAUAGGACA 24 upstream 1175 (SEQ ID NO: 1982) CEP290- +UAAUGCUGGAGAGGAUAGGACA (SEQ 22 upstream 1176 ID NO: 1983) CEP290- +UGCUGGAGAGGAUAGGACA (SEQ ID 19 upstream 1177 NO: 1984) CEP290- −UAUCAUAAGUUACAAUCUGUGAAU 24 upstream 1178 (SEQ ID NO: 1985) CEP290- −UCAUAAGUUACAAUCUGUGAAU (SEQ 22 upstream 1179 ID NO: 1986) CEP290- −UAAGUUACAAUCUGUGAAU (SEQ ID 19 upstream 1180 NO: 1987) CEP290- −UUUAACCAGACAUCUAAGAGAAAA 24 upstream 1181 (SEQ ID NO: 1988) CEP290- −UUAACCAGACAUCUAAGAGAAAA (SEQ 23 upstream 1182 ID NO: 1989) CEP290- −UAACCAGACAUCUAAGAGAAAA (SEQ 22 upstream 1183 ID NO: 1990) CEP290- +UCUAUUUCUGAUGAGGAAG (SEQ ID 19 upstream 1184 NO: 1991) CEP290- +UCUGAUGAGGAAGAUGAACAAAUC 24 upstream 1185 (SEQ ID NO: 1992) CEP290- +UGAUGAGGAAGAUGAACAAAUC (SEQ 22 upstream 1186 ID NO: 1993) CEP290- +UGAGGAAGAUGAACAAAUC (SEQ ID 19 upstream 1187 NO: 1994) CEP290- +UUUUCAUUUACUGAAUGUGUCUCU 24 upstream 1188 (SEQ ID NO: 1995) CEP290- +UUUCAUUUACUGAAUGUGUCUCU (SEQ 23 upstream 1189 ID NO: 1996) CEP290- +UUCAUUUACUGAAUGUGUCUCU (SEQ 22 upstream 1190 ID NO: 1997) CEP290- +UCAUUUACUGAAUGUGUCUCU (SEQ ID 21 upstream 1191 NO: 1998) CEP290- +UUUACUGAAUGUGUCUCU (SEQ ID NO: 18 upstream 1192 1999) CEP290- +UGGGUACAGGGGUAAGAGAAAGGG 24 upstream 1193 (SEQ ID NO: 2000) CEP290- +UACAGGGGUAAGAGAAAGGG (SEQ ID 20 upstream 1194 NO: 2001) CEP290- +UGUGUGUGUGUGUGUGUGUUAUGU 24 upstream 1195 (SEQ ID NO: 2002) CEP290- +UGUGUGUGUGUGUGUGUUAUGU (SEQ 22 upstream 1196 ID NO: 2003) CEP290- +UGUGUGUGUGUGUGUUAUGU (SEQ ID 20 upstream 1197 NO: 1185) CEP290- +UGUGUGUGUGUGUUAUGU (SEQ ID NO: 18 upstream 1198 2005) CEP290- +AUUUACAGAGUGCAUCCAUGGUCC 24 upstream 1199 (SEQ ID NO: 2006) CEP290- +ACAGAGUGCAUCCAUGGUCC (SEQ ID 20 upstream 1200 NO: 1085) CEP290- +AGAGUGCAUCCAUGGUCC (SEQ ID NO: 18 upstream 1201 2008) CEP290- −ACUUGAACUCUAUACCUUUUA (SEQ ID 21 upstream 1202 NO: 2009) CEP290- +AGCUAAAUCAUGCAAGUGACCU (SEQ 22 upstream 1203 ID NO: 2010) CEP290- +AAAUCAUGCAAGUGACCU (SEQ ID NO: 18 upstream 1204 2011) CEP290- +AUCCAUAAGCCUCUAUUUCUGAUG 24 upstream 1205 (SEQ ID NO: 2012) CEP290- +AUAAGCCUCUAUUUCUGAUG (SEQ ID 20 upstream 1206 NO: 723) CEP290- +AAGCCUCUAUUUCUGAUG (SEQ ID NO: 18 upstream 1207 2014) CEP290- +AGAAUAGUUUGUUCUGGGUA (SEQ ID 20 upstream 1208 NO: 2015) CEP290- +AAUAGUUUGUUCUGGGUA (SEQ ID NO: 18 upstream 1209 2016) CEP290- +AGGAGAAUGAUCUAGAUAAUCAUU 24 upstream 1210 (SEQ ID NO: 2017) CEP290- +AGAAUGAUCUAGAUAAUCAUU (SEQ ID 21 upstream 1211 NO: 2018) CEP290- +AAUGAUCUAGAUAAUCAUU (SEQ ID 19 upstream 1212 NO: 2019) CEP290- +AUGAUCUAGAUAAUCAUU (SEQ ID NO: 18 upstream 1213 2020) CEP290- +AAUGCUGGAGAGGAUAGGA (SEQ ID 19 upstream 1214 NO: 2021) CEP290- +AUGCUGGAGAGGAUAGGA (SEQ ID NO: 18 upstream 1215 2022) CEP290- +AAAAUCCAUAAGCCUCUAUUUCUG 24 upstream 1216 (SEQ ID NO: 2023) CEP290- +AAAUCCAUAAGCCUCUAUUUCUG (SEQ 23 upstream 1217 ID NO: 2024) CEP290- +AAUCCAUAAGCCUCUAUUUCUG (SEQ 22 upstream 1218 ID NO: 2025) CEP290- +AUCCAUAAGCCUCUAUUUCUG (SEQ ID 21 upstream 1219 NO: 2026) CEP290- −AAACAGGUAGAAUAUUGUAAUCA 23 upstream 1220 (SEQ ID NO: 2027) CEP290- −AACAGGUAGAAUAUUGUAAUCA (SEQ 22 upstream 1221 ID NO: 2028) CEP290- −ACAGGUAGAAUAUUGUAAUCA (SEQ ID 21 upstream 1222 NO: 2029) CEP290- −AGGUAGAAUAUUGUAAUCA (SEQ ID 19 upstream 1223 NO: 2030) CEP290- +AAGGAACAAAAGCCAGGGACCA (SEQ 22 upstream 1224 ID NO: 2031) CEP290- +AGGAACAAAAGCCAGGGACCA (SEQ ID 21 upstream 1225 NO: 2032) CEP290- +AACAAAAGCCAGGGACCA (SEQ ID NO: 18 upstream 1226 2033) CEP290- −AGGUAGAAUAUUGUAAUCAAAGGA 24 upstream 1227 (SEQ ID NO: 2034) CEP290- −AGAAUAUUGUAAUCAAAGGA (SEQ ID 20 upstream 1228 NO: 1089) CEP290- −AAUAUUGUAAUCAAAGGA (SEQ ID NO: 18 upstream 1229 2036) CEP290- −AGUCAUGUUUAUCAAUAUUAUU (SEQ 22 upstream 1230 ID NO: 2037) CEP290- −AUGUUUAUCAAUAUUAUU (SEQ ID NO: 18 upstream 1231 2038) CEP290- −AACCAGACAUCUAAGAGAAA (SEQ ID 20 upstream 1232 NO: 2039) CEP290- −ACCAGACAUCUAAGAGAAA (SEQ ID 19 upstream 1233 NO: 2040) CEP290- −AUUCUUAUCUAAGAUCCUUUCA (SEQ 22 upstream 1234 ID NO: 2041) CEP290- −AAACAGGUAGAAUAUUGUAAUCAA 24 upstream 1235 (SEQ ID NO: 2042) CEP290- −AACAGGUAGAAUAUUGUAAUCAA 23 upstream 1236 (SEQ ID NO: 2043) CEP290- −ACAGGUAGAAUAUUGUAAUCAA (SEQ 22 upstream 1237 ID NO: 2044) CEP290- −AGGUAGAAUAUUGUAAUCAA (SEQ ID 20 upstream 1238 NO: 1101) CEP290- +AUGAGGAAGAUGAACAAAU (SEQ ID 19 upstream 1239 NO: 2046) CEP290- +AGAGGAUAGGACAGAGGAC (SEQ ID 19 upstream 1240 NO: 2047) CEP290- +CAGAGUGCAUCCAUGGUCC (SEQ ID 19 upstream 1241 NO: 2048) CEP290- +CUUGCCUAGGACUUUCUAAUGCUG 24 upstream 1242 (SEQ ID NO: 2049) CEP290- +CCUAGGACUUUCUAAUGCUG (SEQ ID 20 upstream 1243 NO: 858) CEP290- +CUAGGACUUUCUAAUGCUG (SEQ ID 19 upstream 1244 NO: 2051) CEP290- −CCACUUGAACUCUAUACCUUUUA (SEQ 23 upstream 1245 ID NO: 2052) CEP290- −CACUUGAACUCUAUACCUUUUA (SEQ 22 upstream 1246 ID NO: 2053) CEP290- −CUUGAACUCUAUACCUUUUA (SEQ ID 20 upstream 1247 NO: 2054) CEP290- +CAGCUAAAUCAUGCAAGUGACCU (SEQ 23 upstream 1248 ID NO: 2055) CEP290- +CUAAAUCAUGCAAGUGACCU (SEQ ID 20 upstream 1249 NO: 2056) CEP290- +CUCUUGCCUAGGACUUUCUAAUG (SEQ 23 upstream 1250 ID NO: 2057) CEP290- +CUUGCCUAGGACUUUCUAAUG (SEQ ID 21 upstream 1251 NO: 2058) CEP290- +CCAUAAGCCUCUAUUUCUGAUG (SEQ 22 upstream 1252 ID NO: 2059) CEP290- +CAUAAGCCUCUAUUUCUGAUG (SEQ ID 21 upstream 1253 NO: 2060) CEP290- +CUAAUGCUGGAGAGGAUAGGA (SEQ ID 21 upstream 1254 NO: 2061) CEP290- +CCAUAAGCCUCUAUUUCUG (SEQ ID 19 upstream 1255 NO: 2062) CEP290- +CAUAAGCCUCUAUUUCUG (SEQ ID NO: 18 upstream 1256 2063) CEP290- −CAGGUAGAAUAUUGUAAUCA (SEQ ID 20 upstream 1257 NO: 2064) CEP290- −CUUUCUGCUGCUUUUGCCAAA (SEQ ID 21 upstream 1258 NO: 2065) CEP290- +CCAAGGAACAAAAGCCAGGGACCA 24 upstream 1259 (SEQ ID NO: 2066) CEP290- +CAAGGAACAAAAGCCAGGGACCA (SEQ 23 upstream 1260 ID NO: 2067) CEP290- +CUCUUAGAUGUCUGGUUAA (SEQ ID 19 upstream 1261 NO: 2068) CEP290- −CAUGUUUAUCAAUAUUAUU (SEQ ID 19 upstream 1262 NO: 2069) CEP290- −CCAGACAUCUAAGAGAAA (SEQ ID NO: 18 upstream 1263 2070) CEP290- −CUUAUCUAAGAUCCUUUCA (SEQ ID 19 upstream 1264 NO: 2071) CEP290- −CAGGUAGAAUAUUGUAAUCAA (SEQ ID 21 upstream 1265 NO: 2072) CEP290- +CUGAUGAGGAAGAUGAACAAAU (SEQ 22 upstream 1266 ID NO: 2073) CEP290- +CUGGAGAGGAUAGGACAGAGGAC 23 upstream 1267 (SEQ ID NO: 2074) CEP290- −CAUCUUCCUCAUCAGAAA (SEQ ID NO: 18 upstream 1268 2075) CEP290- +GCCUAGGACUUUCUAAUGCUG (SEQ ID 21 upstream 1269 NO: 2076) CEP290- −GCCACUUGAACUCUAUACCUUUUA 24 upstream 1270 (SEQ ID NO: 2077) CEP290- +GCUAAAUCAUGCAAGUGACCU (SEQ ID 21 upstream 1271 NO: 2078) CEP290- +GCCUAGGACUUUCUAAUG (SEQ ID NO: 18 upstream 1272 2079) CEP290- +GGGAGAAUAGUUUGUUCUGGGUA 23 upstream 1273 (SEQ ID NO: 2080) CEP290- +GGAGAAUAGUUUGUUCUGGGUA (SEQ 22 upstream 1274 ID NO: 2081) CEP290- +GAGAAUAGUUUGUUCUGGGUA (SEQ 21 upstream 1275 ID NO: 2082) CEP290- +GAAUAGUUUGUUCUGGGUA (SEQ ID 19 upstream 1276 NO: 2083) CEP290- +GGAGAAUGAUCUAGAUAAUCAUU 23 upstream 1277 (SEQ ID NO: 2084) CEP290- +GAGAAUGAUCUAGAUAAUCAUU (SEQ 22 upstream 1278 ID NO: 2085) CEP290- +GAAUGAUCUAGAUAAUCAUU (SEQ ID 20 upstream 1279 NO: 2086) CEP290- −GAAACAGGUAGAAUAUUGUAAUCA 24 upstream 1280 (SEQ ID NO: 2087) CEP290- −GGUAGAAUAUUGUAAUCA (SEQ ID NO: 18 upstream 1281 2088) CEP290- −GCUUUCUGCUGCUUUUGCCAAA (SEQ 22 upstream 1282 ID NO: 2089) CEP290- +GGAACAAAAGCCAGGGACCA (SEQ ID 20 upstream 1283 NO: 484) CEP290- +GAACAAAAGCCAGGGACCA (SEQ ID 19 upstream 1284 NO: 2090) CEP290- −GGUAGAAUAUUGUAAUCAAAGGA 23 upstream 1285 (SEQ ID NO: 2091) CEP290- −GUAGAAUAUUGUAAUCAAAGGA (SEQ 22 upstream 1286 ID NO: 2092) CEP290- −GAAUAUUGUAAUCAAAGGA (SEQ ID 19 upstream 1287 NO: 2093) CEP290- −GAGUCAUGUUUAUCAAUAUUAUU 23 upstream 1288 (SEQ ID NO: 2094) CEP290- −GUCAUGUUUAUCAAUAUUAUU (SEQ ID 21 upstream 1289 NO: 2095) CEP290- −GGUAGAAUAUUGUAAUCAA (SEQ ID 19 upstream 1290 NO: 2096) CEP290- −GUAGAAUAUUGUAAUCAA (SEQ ID NO: 18 upstream 1291 2097) CEP290- +GAUGAGGAAGAUGAACAAAU (SEQ ID 20 upstream 1292 NO: 773) CEP290- +GCUGGAGAGGAUAGGACAGAGGAC 24 upstream 1293 (SEQ ID NO: 2099) CEP290- +GGAGAGGAUAGGACAGAGGAC (SEQ ID 21 upstream 1294 NO: 2100) CEP290- +GAGAGGAUAGGACAGAGGAC (SEQ ID 20 upstream 1295 NO: 772) CEP290- +GAGGAUAGGACAGAGGAC (SEQ ID NO: 18 upstream 1296 2102) CEP290- −GUUCAUCUUCCUCAUCAGAAA (SEQ ID 21 upstream 1297 NO: 2103) CEP290- +UUUACAGAGUGCAUCCAUGGUCC (SEQ 23 upstream 1298 ID NO: 2104) CEP290- +UUACAGAGUGCAUCCAUGGUCC (SEQ 22 upstream 1299 ID NO: 2105) CEP290- +UACAGAGUGCAUCCAUGGUCC (SEQ ID 21 upstream 1300 NO: 2106) CEP290- +UUGCCUAGGACUUUCUAAUGCUG (SEQ 23 upstream 1301 ID NO: 2107) CEP290- +UGCCUAGGACUUUCUAAUGCUG (SEQ 22 upstream 1302 ID NO: 2108) CEP290- +UAGGACUUUCUAAUGCUG (SEQ ID NO: 18 upstream 1303 2109) CEP290- −UUGAACUCUAUACCUUUUA (SEQ ID 19 upstream 1304 NO: 2110) CEP290- −UGAACUCUAUACCUUUUA (SEQ ID NO: 18 upstream 1305 2111) CEP290- +UCAGCUAAAUCAUGCAAGUGACCU 24 upstream 1306 (SEQ ID NO: 2112) CEP290- +UAAAUCAUGCAAGUGACCU (SEQ ID 19 upstream 1307 NO: 2113) CEP290- +UCUCUUGCCUAGGACUUUCUAAUG 24 upstream 1308 (SEQ ID NO: 2114) CEP290- +UCUUGCCUAGGACUUUCUAAUG (SEQ 22 upstream 1309 ID NO: 2115) CEP290- +UUGCCUAGGACUUUCUAAUG (SEQ ID 20 upstream 1310 NO: 906) CEP290- +UGCCUAGGACUUUCUAAUG (SEQ ID 19 upstream 1311 NO: 2117) CEP290- +UCCAUAAGCCUCUAUUUCUGAUG (SEQ 23 upstream 1312 ID NO: 2118) CEP290- +UAAGCCUCUAUUUCUGAUG (SEQ ID 19 upstream 1313 NO: 2119) CEP290- +UGGGAGAAUAGUUUGUUCUGGGUA 24 upstream 1314 (SEQ ID NO: 2120) CEP290- +UUUCUAAUGCUGGAGAGGAUAGGA 24 upstream 1315 (SEQ ID NO: 2121) CEP290- +UUCUAAUGCUGGAGAGGAUAGGA 23 upstream 1316 (SEQ ID NO: 2122) CEP290- +UCUAAUGCUGGAGAGGAUAGGA (SEQ 22 upstream 1317 ID NO: 2123) CEP290- +UAAUGCUGGAGAGGAUAGGA (SEQ ID 20 upstream 1318 NO: 873) CEP290- +UCCAUAAGCCUCUAUUUCUG (SEQ ID 20 upstream 1319 NO: 886) CEP290- −UUGCUUUCUGCUGCUUUUGCCAAA 24 upstream 1320 (SEQ ID NO: 2126) CEP290- −UGCUUUCUGCUGCUUUUGCCAAA (SEQ 23 upstream 1321 ID NO: 2127) CEP290- −UUUCUGCUGCUUUUGCCAAA (SEQ ID 20 upstream 1322 NO: 907) CEP290- −UUCUGCUGCUUUUGCCAAA (SEQ ID 19 upstream 1323 NO: 2129) CEP290- −UCUGCUGCUUUUGCCAAA (SEQ ID NO: 18 upstream 1324 2130) CEP290- −UAGAAUAUUGUAAUCAAAGGA (SEQ 21 upstream 1325 ID NO: 2131) CEP290- +UUUUUCUCUUAGAUGUCUGGUUAA 24 upstream 1326 (SEQ ID NO: 2132) CEP290- +UUUUCUCUUAGAUGUCUGGUUAA 23 upstream 1327 (SEQ ID NO: 2133) CEP290- +UUUCUCUUAGAUGUCUGGUUAA (SEQ 22 upstream 1328 ID NO: 2134) CEP290- +UUCUCUUAGAUGUCUGGUUAA (SEQ ID 21 upstream 1329 NO: 2135) CEP290- +UCUCUUAGAUGUCUGGUUAA (SEQ ID 20 upstream 1330 NO: 2136) CEP290- +UCUUAGAUGUCUGGUUAA (SEQ ID NO: 18 upstream 1331 2137) CEP290- −UGAGUCAUGUUUAUCAAUAUUAUU 24 upstream 1332 (SEQ ID NO: 2138) CEP290- −UCAUGUUUAUCAAUAUUAUU (SEQ ID 20 upstream 1333 NO: 884) CEP290- −UUUUAACCAGACAUCUAAGAGAAA 24 upstream 1334 (SEQ ID NO: 2140) CEP290- −UUUAACCAGACAUCUAAGAGAAA (SEQ 23 upstream 1335 ID NO: 2141) CEP290- −UUAACCAGACAUCUAAGAGAAA (SEQ 22 upstream 1336 ID NO: 2142) CEP290- −UAACCAGACAUCUAAGAGAAA (SEQ ID 21 upstream 1337 NO: 2143) CEP290- −UUAUUCUUAUCUAAGAUCCUUUCA 24 upstream 1338 (SEQ ID NO: 2144) CEP290- −UAUUCUUAUCUAAGAUCCUUUCA (SEQ 23 upstream 1339 ID NO: 2145) CEP290- −UUCUUAUCUAAGAUCCUUUCA (SEQ ID 21 upstream 1340 NO: 2146) CEP290- −UCUUAUCUAAGAUCCUUUCA (SEQ ID 20 upstream 1341 NO: 892) CEP290- −UUAUCUAAGAUCCUUUCA (SEQ ID NO: 18 upstream 1342 2148) CEP290- +UUCUGAUGAGGAAGAUGAACAAAU 24 upstream 1343 (SEQ ID NO: 2149) CEP290- +UCUGAUGAGGAAGAUGAACAAAU 23 upstream 1344 (SEQ ID NO: 2150) CEP290- +UGAUGAGGAAGAUGAACAAAU (SEQ 21 upstream 1345 ID NO: 2151) CEP290- +UGAGGAAGAUGAACAAAU (SEQ ID NO: 18 upstream 1346 2152) CEP290- +UGGAGAGGAUAGGACAGAGGAC (SEQ 22 upstream 1347 ID NO: 2153) CEP290- −UUUGUUCAUCUUCCUCAUCAGAAA 24 upstream 1348 (SEQ ID NO: 2154) CEP290- −UUGUUCAUCUUCCUCAUCAGAAA (SEQ 23 upstream 1349 ID NO: 2155) CEP290- −UGUUCAUCUUCCUCAUCAGAAA (SEQ 22 upstream 1350 ID NO: 2156) CEP290- −UUCAUCUUCCUCAUCAGAAA (SEQ ID 20 upstream 1351 NO: 905) CEP290- −UCAUCUUCCUCAUCAGAAA (SEQ ID 19 upstream 1352 NO: 2158) CEP290- −ACUUACCUCAUGUCAUCUAGAGC (SEQ 23 downstream 1353 ID NO: 2159) CEP290- −ACCUCAUGUCAUCUAGAGC (SEQ ID 19 downstream 1354 NO: 2160) CEP290- +ACAGUUUUUAAGGCGGGGAGUCAC 24 downstream 1355 (SEQ ID NO: 2161) CEP290- +AGUUUUUAAGGCGGGGAGUCAC (SEQ 22 downstream 1356 ID NO: 2162) CEP290- −ACAGAGUUCAAGCUAAUAC (SEQ ID 19 downstream 1357 NO: 2163) CEP290- +AUUAGCUUGAACUCUGUGCCAAAC 24 downstream 1358 (SEQ ID NO: 2164) CEP290- +AGCUUGAACUCUGUGCCAAAC (SEQ ID 21 downstream 1359 NO: 2165) CEP290- −AUGUGGUGUCAAAUAUGGUGCU (SEQ 22 downstream 1360 ID NO: 2166) CEP290- −AUGUGGUGUCAAAUAUGGUGCUU 23 downstream 1361 (SEQ ID NO: 2167) CEP290- +AGAUGACAUGAGGUAAGU (SEQ ID NO: 18 downstream 1362 2168) CEP290- −AAUACAUGAGAGUGAUUAGUGG (SEQ 22 downstream 1363 ID NO: 2169) CEP290- −AUACAUGAGAGUGAUUAGUGG (SEQ 21 downstream 1364 ID NO: 2170) CEP290- −ACAUGAGAGUGAUUAGUGG (SEQ ID 19 downstream 1365 NO: 2171) CEP290-16 +AAGACACUGCCAAUAGGGAUAGGU 24 downstream (SEQ ID NO: 1042) CEP290- +AGACACUGCCAAUAGGGAUAGGU (SEQ 23 downstream 1366 ID NO: 1043) CEP290- +ACACUGCCAAUAGGGAUAGGU (SEQ ID 21 downstream 1367 NO: 1044) CEP290-510 +ACUGCCAAUAGGGAUAGGU (SEQ ID 19 downstream NO: 1045) CEP290- −AAAGGUUCAUGAGACUAGAGGUC 23 downstream 1368 (SEQ ID NO: 2176) CEP290- −AAGGUUCAUGAGACUAGAGGUC (SEQ 22 downstream 1369 ID NO: 2177) CEP290- −AGGUUCAUGAGACUAGAGGUC (SEQ ID 21 downstream 1370 NO: 2178) CEP290- +AAACAGGAGAUACUCAACACA (SEQ ID 21 downstream 1371 NO: 2179) CEP290- +AACAGGAGAUACUCAACACA (SEQ ID 20 downstream 1372 NO: 810) CEP290- +ACAGGAGAUACUCAACACA (SEQ ID 19 downstream 1373 NO: 2181) CEP290- +AGCACGUACAAAAGAACAUACAU (SEQ 23 downstream 1374 ID NO: 2182) CEP290- +ACGUACAAAAGAACAUACAU (SEQ ID 20 downstream 1375 NO: 817) CEP290- +AGUAAGGAGGAUGUAAGAC (SEQ ID 19 downstream 1376 NO: 2184) CEP290- +AGCUUUUGACAGUUUUUAAGG (SEQ ID 21 downstream 1377 NO: 2185) CEP290- −ACGUGCUCUUUUCUAUAUAU (SEQ ID 20 downstream 1378 NO: 622) CEP290- +AAAUUCACUGAGCAAAACAACUGG 24 downstream 1379 (SEQ ID NO: 2186) CEP290- +AAUUCACUGAGCAAAACAACUGG (SEQ 23 downstream 1380 ID NO: 2187) CEP290- +AUUCACUGAGCAAAACAACUGG (SEQ 22 downstream 1381 ID NO: 2188) CEP290- +ACUGAGCAAAACAACUGG (SEQ ID NO: 18 downstream 1382 2189) CEP290- +AACAAGUUUUGAAACAGGAA (SEQ ID 20 downstream 1383 NO: 809) CEP290- +ACAAGUUUUGAAACAGGAA (SEQ ID 19 downstream 1384 NO: 2191) CEP290- +AAUGCCUGAACAAGUUUUGAAA (SEQ 22 downstream 1385 ID NO: 2192) CEP290- +AUGCCUGAACAAGUUUUGAAA (SEQ ID 21 downstream 1386 NO: 2193) CEP290- +AUUCACUGAGCAAAACAACUGGAA 24 downstream 1387 (SEQ ID NO: 2194) CEP290- +ACUGAGCAAAACAACUGGAA (SEQ ID 20 downstream 1388 NO: 819) CEP290- +AAAAAGGUAAUGCCUGAACAAGUU 24 downstream 1389 (SEQ ID NO: 2196) CEP290- +AAAAGGUAAUGCCUGAACAAGUU 23 downstream 1390 (SEQ ID NO: 2197) CEP290- +AAAGGUAAUGCCUGAACAAGUU (SEQ 22 downstream 1391 ID NO: 2198) CEP290- +AAGGUAAUGCCUGAACAAGUU (SEQ ID 21 downstream 1392 NO: 2199) CEP290- +AGGUAAUGCCUGAACAAGUU (SEQ ID 20 downstream 1393 NO: 828) CEP290- −ACGUGCUCUUUUCUAUAUA (SEQ ID 19 downstream 1394 NO: 2201) CEP290- +AUUAUCUAUUCCAUUCUUCACAC (SEQ 23 downstream 1395 ID NO: 2202) CEP290- +AUCUAUUCCAUUCUUCACAC (SEQ ID 20 downstream 1396 NO: 2203) CEP290- +AAGAGAGAAAUGGUUCCCUAUAUA 24 downstream 1397 (SEQ ID NO: 2204) CEP290- +AGAGAGAAAUGGUUCCCUAUAUA 23 downstream 1398 (SEQ ID NO: 2205) CEP290- +AGAGAAAUGGUUCCCUAUAUA (SEQ ID 21 downstream 1399 NO: 2206) CEP290- +AGAAAUGGUUCCCUAUAUA (SEQ ID 19 downstream 1400 NO: 2207) CEP290- −AGGAAAUUAUUGUUGCUUU (SEQ ID 19 downstream 1401 NO: 2208) CEP290- +ACUGAGCAAAACAACUGGAAGA (SEQ 22 downstream 1402 ID NO: 2209) CEP290- +AGCAAAACAACUGGAAGA (SEQ ID NO: 18 downstream 1403 2210) CEP290- +AUACAUAAGAAAGAACACUGUGGU 24 downstream 1404 (SEQ ID NO: 2211) CEP290- +ACAUAAGAAAGAACACUGUGGU (SEQ 22 downstream 1405 ID NO: 2212) CEP290- +AUAAGAAAGAACACUGUGGU (SEQ ID 20 downstream 1406 NO: 829) CEP290- +AAGAAAGAACACUGUGGU (SEQ ID NO: 18 downstream 1407 2214) CEP290- −AAGAAUGGAAUAGAUAAU (SEQ ID NO: 18 downstream 1408 2215) CEP290- +AAGGAGGAUGUAAGACUGGAGA (SEQ 22 downstream 1409 ID NO: 2216) CEP290- +AGGAGGAUGUAAGACUGGAGA (SEQ 21 downstream 1410 ID NO: 2217) CEP290- +AGGAUGUAAGACUGGAGA (SEQ ID NO: 18 downstream 1411 2218) CEP290- −AAAAACUUGAAAUUUGAUAGUAG 23 downstream 1412 (SEQ ID NO: 2219) CEP290- −AAAACUUGAAAUUUGAUAGUAG (SEQ 22 downstream 1413 ID NO: 2220) CEP290- −AAACUUGAAAUUUGAUAGUAG (SEQ 21 downstream 1414 ID NO: 2221) CEP290- −AACUUGAAAUUUGAUAGUAG (SEQ ID 20 downstream 1415 NO: 2222) CEP290- −ACUUGAAAUUUGAUAGUAG (SEQ ID 19 downstream 1416 NO: 2223) CEP290- −ACAUAUCUGUCUUCCUUA (SEQ ID NO: 18 downstream 1417 2224) CEP290- +AUUAAAAAAAGUAUGCUU (SEQ ID NO: 18 downstream 1418 2225) CEP290- +AUAUCAAAAGACUUAUAUUCCAUU 24 downstream 1419 (SEQ ID NO: 2226) CEP290- +AUCAAAAGACUUAUAUUCCAUU (SEQ 22 downstream 1420 ID NO: 2227) CEP290- +AAAAGACUUAUAUUCCAUU (SEQ ID 19 downstream 1421 NO: 2228) CEP290- +AAAGACUUAUAUUCCAUU (SEQ ID NO: 18 downstream 1422 2229) CEP290- −AAAAUCAGAUUUCAUGUGUGAAGA 24 downstream 1423 (SEQ ID NO: 2230) CEP290- −AAAUCAGAUUUCAUGUGUGAAGA 23 downstream 1424 (SEQ ID NO: 2231) CEP290- −AAUCAGAUUUCAUGUGUGAAGA (SEQ 22 downstream 1425 ID NO: 2232) CEP290- −AUCAGAUUUCAUGUGUGAAGA (SEQ ID 21 downstream 1426 NO: 2233) CEP290- −AGAUUUCAUGUGUGAAGA (SEQ ID NO: 18 downstream 1427 2234) CEP290- −AAUGGAAUAUAAGUCUUUUGAUAU 24 downstream 1428 (SEQ ID NO: 2235) CEP290- −AUGGAAUAUAAGUCUUUUGAUAU 23 downstream 1429 (SEQ ID NO: 2236) CEP290- −AAUAUAAGUCUUUUGAUAU (SEQ ID 19 downstream 1430 NO: 2237) CEP290- −AUAUAAGUCUUUUGAUAU (SEQ ID NO: 18 downstream 1431 2238) CEP290- −AAGAAUGGAAUAGAUAAUA (SEQ ID 19 downstream 1432 NO: 2239) CEP290- −AGAAUGGAAUAGAUAAUA (SEQ ID NO: 18 downstream 1433 2240) CEP290- −AAAACUGGAUGGGUAAUAAAGCAA 24 downstream 1434 (SEQ ID NO: 2241) CEP290- −AAACUGGAUGGGUAAUAAAGCAA 23 downstream 1435 (SEQ ID NO: 2242) CEP290- −AACUGGAUGGGUAAUAAAGCAA (SEQ 22 downstream 1436 ID NO: 2243) CEP290- −ACUGGAUGGGUAAUAAAGCAA (SEQ ID 21 downstream 1437 NO: 2244) CEP290- +AUAGAAAUUCACUGAGCAAAACAA 24 downstream 1438 (SEQ ID NO: 2245) CEP290- +AGAAAUUCACUGAGCAAAACAA (SEQ 22 downstream 1439 ID NO: 2246) CEP290- +AAAUUCACUGAGCAAAACAA (SEQ ID 20 downstream 1440 NO: 808) CEP290- +AAUUCACUGAGCAAAACAA (SEQ ID 19 downstream 1441 NO: 2248) CEP290- +AUUCACUGAGCAAAACAA (SEQ ID NO: 18 downstream 1442 2249) CEP290- +AGGAUGUAAGACUGGAGAUAGAGA 24 downstream 1443 (SEQ ID NO: 2250) CEP290- +AUGUAAGACUGGAGAUAGAGA (SEQ 21 downstream 1444 ID NO: 2251) CEP290- −AAAUUUGAUAGUAGAAGAAAA (SEQ 21 downstream 1445 ID NO: 2252) CEP290- −AAUUUGAUAGUAGAAGAAAA (SEQ ID 20 downstream 1446 NO: 2253) CEP290- −AUUUGAUAGUAGAAGAAAA (SEQ ID 19 downstream 1447 NO: 2254) CEP290- +AAAAUAAAACUAAGACACUGCCAA 24 downstream 1448 (SEQ ID NO: 1036) CEP290- +AAAUAAAACUAAGACACUGCCAA (SEQ 23 downstream 1449 ID NO: 1037) CEP290- +AAUAAAACUAAGACACUGCCAA (SEQ 22 downstream 1450 ID NO: 1038) CEP290- +AUAAAACUAAGACACUGCCAA (SEQ ID 21 downstream 1451 NO: 1039) CEP290- +AAAACUAAGACACUGCCAA (SEQ ID 19 downstream 1452 NO: 1040) CEP290- +AAACUAAGACACUGCCAA (SEQ ID NO: 18 downstream 1453 1041) CEP290- −AAUAAAGCAAAAGAAAAAC (SEQ ID 19 downstream 1454 NO: 2261) CEP290- −AUAAAGCAAAAGAAAAAC (SEQ ID NO: 18 downstream 1455 2262) CEP290- −AUUCUUUUUUUGUUGUUUUUUUUU 24 downstream 1456 (SEQ ID NO: 2263) CEP290- +ACUCCAGCCUGGGCAACACA (SEQ ID 20 downstream 1457 NO: 2264) CEP290- −CUUACCUCAUGUCAUCUAGAGC (SEQ 22 downstream 1458 ID NO: 2265) CEP290- −CCUCAUGUCAUCUAGAGC (SEQ ID NO: 18 downstream 1459 2266) CEP290- +CAGUUUUUAAGGCGGGGAGUCAC (SEQ 23 downstream 1460 ID NO: 2267) CEP290- −CACAGAGUUCAAGCUAAUAC (SEQ ID 20 downstream 1461 NO: 845) CEP290- −CAGAGUUCAAGCUAAUAC (SEQ ID NO: 18 downstream 1462 2269) CEP290- +CUUGAACUCUGUGCCAAAC (SEQ ID 19 downstream 1463 NO: 2270) CEP290- −CAUGUGGUGUCAAAUAUGGUGCU 23 downstream 1464 (SEQ ID NO: 2271) CEP290- −CAUGUGGUGUCAAAUAUGGUGCUU 24 downstream 1465 (SEQ ID NO: 2272) CEP290- +CUCUAGAUGACAUGAGGUAAGU (SEQ 22 downstream 1466 ID NO: 2273) CEP290- +CUAGAUGACAUGAGGUAAGU (SEQ ID 20 downstream 1467 NO: 671) CEP290- −CUAAUACAUGAGAGUGAUUAGUGG 24 downstream 1468 (SEQ ID NO: 2275) CEP290- −CAUGAGAGUGAUUAGUGG (SEQ ID NO: 18 downstream 1469 2276) CEP290-509 +CACUGCCAAUAGGGAUAGGU (SEQ ID 20 downstream NO: 613) CEP290-511 +CUGCCAAUAGGGAUAGGU (SEQ ID NO: 18 downstream 1046) CEP290- +CCAAACAGGAGAUACUCAACACA (SEQ 23 downstream 1470 ID NO: 2278) CEP290- +CAAACAGGAGAUACUCAACACA (SEQ 22 downstream 1471 ID NO: 2279) CEP290- +CAGGAGAUACUCAACACA (SEQ ID NO: 18 downstream 1472 2280) CEP290- +CACGUACAAAAGAACAUACAU (SEQ ID 21 downstream 1473 NO: 2281) CEP290- +CGUACAAAAGAACAUACAU (SEQ ID 19 downstream 1474 NO: 2282) CEP290- +CAGUAAGGAGGAUGUAAGAC (SEQ ID 20 downstream 1475 NO: 676) CEP290- +CUUUUGACAGUUUUUAAGG (SEQ ID 19 downstream 1476 NO: 2284) CEP290- −CGUGCUCUUUUCUAUAUAU (SEQ ID 19 downstream 1477 NO: 2285) CEP290- +CACUGAGCAAAACAACUGG (SEQ ID 19 downstream 1478 NO: 2286) CEP290- +CCUGAACAAGUUUUGAAACAGGAA 24 downstream 1479 (SEQ ID NO: 2287) CEP290- +CUGAACAAGUUUUGAAACAGGAA 23 downstream 1480 (SEQ ID NO: 2288) CEP290- +CAAGUUUUGAAACAGGAA (SEQ ID NO: 18 downstream 1481 2289) CEP290- +CCUGAACAAGUUUUGAAA (SEQ ID NO: 18 downstream 1482 2290) CEP290- +CACUGAGCAAAACAACUGGAA (SEQ ID 21 downstream 1483 NO: 2291) CEP290- +CUGAGCAAAACAACUGGAA (SEQ ID 19 downstream 1484 NO: 2292) CEP290- −CGUGCUCUUUUCUAUAUA (SEQ ID NO: 18 downstream 1485 2293) CEP290- +CUAUUCCAUUCUUCACAC (SEQ ID NO: 18 downstream 1486 2294) CEP290- −CUUAGGAAAUUAUUGUUGCUUU (SEQ 22 downstream 1487 ID NO: 2295) CEP290- −CUUUUUGAGAGGUAAAGGUUC (SEQ ID 21 downstream 1488 NO: 2296) CEP290- +CACUGAGCAAAACAACUGGAAGA (SEQ 23 downstream 1489 ID NO: 2297) CEP290- +CUGAGCAAAACAACUGGAAGA (SEQ ID 21 downstream 1490 NO: 2298) CEP290- +CAUAAGAAAGAACACUGUGGU (SEQ ID 21 downstream 1491 NO: 2299) CEP290- −CUUGAAAUUUGAUAGUAG (SEQ ID NO: 18 downstream 1492 2300) CEP290- +CCAUUAAAAAAAGUAUGCUU (SEQ ID 20 downstream 1493 NO: 857) CEP290- +CAUUAAAAAAAGUAUGCUU (SEQ ID 19 downstream 1494 NO: 2302) CEP290- +CAAAAGACUUAUAUUCCAUU (SEQ ID 20 downstream 1495 NO: 842) CEP290- −CAGAUUUCAUGUGUGAAGA (SEQ ID 19 downstream 1496 NO: 2304) CEP290- −CUGGAUGGGUAAUAAAGCAA (SEQ ID 20 downstream 1497 NO: 2305) CEP290- −CUUAAGCAUACUUUUUUUUUA (SEQ ID 19 downstream 1498 NO: 2306) CEP290- −CUUUUUUUGUUGUUUUUUUUU (SEQ 21 downstream 1499 ID NO: 2307) CEP290- +CUGCACUCCAGCCUGGGCAACACA 24 downstream 1500 (SEQ ID NO: 2308) CEP290- +CACUCCAGCCUGGGCAACACA (SEQ ID 21 downstream 1501 NO: 2309) CEP290- +CUCCAGCCUGGGCAACACA (SEQ ID 19 downstream 1502 NO: 2310) CEP290- +GUUUUUAAGGCGGGGAGUCAC (SEQ ID 21 downstream 1503 NO: 2311) CEP290-230 −GGCACAGAGUUCAAGCUAAUAC (SEQ 22 downstream ID NO: 2312) CEP290- −GCACAGAGUUCAAGCUAAUAC (SEQ ID 21 downstream 1504 NO: 2313) CEP290- +GCUUGAACUCUGUGCCAAAC (SEQ ID 20 downstream 1505 NO: 461) CEP290-139 −GCAUGUGGUGUCAAAUAUGGUGCU 24 downstream (SEQ ID NO: 2314) CEP290- −GUGGUGUCAAAUAUGGUGCU (SEQ ID 20 downstream 1506 NO: 782) CEP290- −GGUGUCAAAUAUGGUGCU (SEQ ID NO: 18 downstream 1507 2316) CEP290- −GUGGUGUCAAAUAUGGUGCUU (SEQ ID 21 downstream 1508 NO: 2317) CEP290- −GGUGUCAAAUAUGGUGCUU (SEQ ID 19 downstream 1509 NO: 2318) CEP290- −GUGUCAAAUAUGGUGCUU (SEQ ID NO: 18 downstream 1510 2319) CEP290- +GCUCUAGAUGACAUGAGGUAAGU 23 downstream 1511 (SEQ ID NO: 2320) CEP290-11 +GACACUGCCAAUAGGGAUAGGU (SEQ 22 downstream ID NO: 1047) CEP290- −GGUUCAUGAGACUAGAGGUC (SEQ ID 20 downstream 1512 NO: 2322) CEP290- −GUUCAUGAGACUAGAGGUC (SEQ ID 19 downstream 1513 NO: 2323) CEP290- +GCCAAACAGGAGAUACUCAACACA 24 downstream 1514 (SEQ ID NO: 2324) CEP290- +GAGCACGUACAAAAGAACAUACAU 24 downstream 1515 (SEQ ID NO: 2325) CEP290- +GCACGUACAAAAGAACAUACAU (SEQ 22 downstream 1516 ID NO: 2326) CEP290- +GUACAAAAGAACAUACAU (SEQ ID NO: 18 downstream 1517 2327) CEP290- +GUGGCAGUAAGGAGGAUGUAAGAC 24 downstream 1518 (SEQ ID NO: 2328) CEP290- +GGCAGUAAGGAGGAUGUAAGAC (SEQ 22 downstream 1519 ID NO: 2329) CEP290- +GCAGUAAGGAGGAUGUAAGAC (SEQ ID 21 downstream 1520 NO: 2330) CEP290- +GUAAGGAGGAUGUAAGAC (SEQ ID NO: 18 downstream 1521 2331) CEP290- +GGUAGCUUUUGACAGUUUUUAAGG 24 downstream 1522 (SEQ ID NO: 2332) CEP290- +GUAGCUUUUGACAGUUUUUAAGG 23 downstream 1523 (SEQ ID NO: 2333) CEP290- +GCUUUUGACAGUUUUUAAGG (SEQ ID 20 downstream 1524 NO: 482) CEP290- −GUACGUGCUCUUUUCUAUAUAU (SEQ 22 downstream 1525 ID NO: 2334) CEP290- −GUGCUCUUUUCUAUAUAU (SEQ ID NO: 18 downstream 1526 2335) CEP290- +GAACAAGUUUUGAAACAGGAA (SEQ ID 21 downstream 1527 NO: 2336) CEP290- +GUAAUGCCUGAACAAGUUUUGAAA 24 downstream 1528 (SEQ ID NO: 2337) CEP290- +GCCUGAACAAGUUUUGAAA (SEQ ID 19 downstream 1529 NO: 2338) CEP290- +GGUAAUGCCUGAACAAGUU (SEQ ID 19 downstream 1530 NO: 2339) CEP290- +GUAAUGCCUGAACAAGUU (SEQ ID NO: 18 downstream 1531 2340) CEP290- −GUACGUGCUCUUUUCUAUAUA (SEQ ID 21 downstream 1532 NO: 2341) CEP290- +GAGAGAAAUGGUUCCCUAUAUA (SEQ 22 downstream 1533 ID NO: 2342) CEP290- +GAGAAAUGGUUCCCUAUAUA (SEQ ID 20 downstream 1534 NO: 771) CEP290- +GAAAUGGUUCCCUAUAUA (SEQ ID NO: 18 downstream 1535 2344) CEP290- −GCUUAGGAAAUUAUUGUUGCUUU 23 downstream 1536 (SEQ ID NO: 2345) CEP290- −GGAAAUUAUUGUUGCUUU (SEQ ID NO: 18 downstream 1537 2346) CEP290- −GCUUUUUGAGAGGUAAAGGUUC (SEQ 22 downstream 1538 ID NO: 2347) CEP290- +GAGCAAAACAACUGGAAGA (SEQ ID 19 downstream 1539 NO: 2348) CEP290- −GUGUGAAGAAUGGAAUAGAUAAU 23 downstream 1540 (SEQ ID NO: 2349) CEP290- −GUGAAGAAUGGAAUAGAUAAU (SEQ 21 downstream 1541 ID NO: 2350) CEP290- −GAAGAAUGGAAUAGAUAAU (SEQ ID 19 downstream 1542 NO: 2351) CEP290- +GUAAGGAGGAUGUAAGACUGGAGA 24 downstream 1543 (SEQ ID NO: 2352) CEP290- +GGAGGAUGUAAGACUGGAGA (SEQ ID 20 downstream 1544 NO: 779) CEP290- +GAGGAUGUAAGACUGGAGA (SEQ ID 19 downstream 1545 NO: 2354) CEP290- −GAAAAACUUGAAAUUUGAUAGUAG 24 downstream 1546 (SEQ ID NO: 2355) CEP290- −GUGUUUACAUAUCUGUCUUCCUUA 24 downstream 1547 (SEQ ID NO: 2356) CEP290- −GUUUACAUAUCUGUCUUCCUUA (SEQ 22 downstream 1548 ID NO: 2357) CEP290- +GUUCCAUUAAAAAAAGUAUGCUU 23 downstream 1549 (SEQ ID NO: 2358) CEP290- −GGAAUAUAAGUCUUUUGAUAU (SEQ 21 downstream 1550 ID NO: 2359) CEP290- −GAAUAUAAGUCUUUUGAUAU (SEQ ID 20 downstream 1551 NO: 770) CEP290- −GUGUGAAGAAUGGAAUAGAUAAUA 24 downstream 1552 (SEQ ID NO: 2361) CEP290- −GUGAAGAAUGGAAUAGAUAAUA (SEQ 22 downstream 1553 ID NO: 2362) CEP290- −GAAGAAUGGAAUAGAUAAUA (SEQ ID 20 downstream 1554 NO: 467) CEP290- −GGAUGGGUAAUAAAGCAA (SEQ ID NO: 18 downstream 1555 2363) CEP290- +GAAAUUCACUGAGCAAAACAA (SEQ ID 21 downstream 1556 NO: 2364) CEP290- +GGAUGUAAGACUGGAGAUAGAGA 23 downstream 1557 (SEQ ID NO: 2365) CEP290- +GAUGUAAGACUGGAGAUAGAGA (SEQ 22 downstream 1558 ID NO: 2366) CEP290- +GUAAGACUGGAGAUAGAGA (SEQ ID 19 downstream 1559 NO: 2367) CEP290- −GAAAUUUGAUAGUAGAAGAAAA (SEQ 22 downstream 1560 ID NO: 2368) CEP290- −GGGUAAUAAAGCAAAAGAAAAAC 23 downstream 1561 (SEQ ID NO: 2369) CEP290- −GGUAAUAAAGCAAAAGAAAAAC (SEQ 22 downstream 1562 ID NO: 2370) CEP290- −GUAAUAAAGCAAAAGAAAAAC (SEQ ID 21 downstream 1563 NO: 2371) CEP290- +GCACUCCAGCCUGGGCAACACA (SEQ 22 downstream 1564 ID NO: 2372) CEP290- −UACUUACCUCAUGUCAUCUAGAGC 24 downstream 1565 (SEQ ID NO: 2373) CEP290- −UUACCUCAUGUCAUCUAGAGC (SEQ ID 21 downstream 1566 NO: 2374) CEP290- −UACCUCAUGUCAUCUAGAGC (SEQ ID 20 downstream 1567 NO: 876) CEP290- +UUUUUAAGGCGGGGAGUCAC (SEQ ID 20 downstream 1568 NO: 909) CEP290- +UUUUAAGGCGGGGAGUCAC (SEQ ID 19 downstream 1569 NO: 2377) CEP290- +UUUAAGGCGGGGAGUCAC (SEQ ID NO: 18 downstream 1570 2378) CEP290- −UUGGCACAGAGUUCAAGCUAAUAC 24 downstream 1571 (SEQ ID NO: 2379) CEP290- −UGGCACAGAGUUCAAGCUAAUAC (SEQ 23 downstream 1572 ID NO: 2380) CEP290- +UUAGCUUGAACUCUGUGCCAAAC (SEQ 23 downstream 1573 ID NO: 2381) CEP290- +UAGCUUGAACUCUGUGCCAAAC (SEQ 22 downstream 1574 ID NO: 2382) CEP290- +UUGAACUCUGUGCCAAAC (SEQ ID NO: 18 downstream 1575 2383) CEP290- −UGUGGUGUCAAAUAUGGUGCU (SEQ ID 21 downstream 1576 NO: 2384) CEP290- −UGGUGUCAAAUAUGGUGCU (SEQ ID 19 downstream 1577 NO: 2385) CEP290- −UGUGGUGUCAAAUAUGGUGCUU (SEQ 22 downstream 1578 ID NO: 2386) CEP290- −UGGUGUCAAAUAUGGUGCUU (SEQ ID 20 downstream 1579 NO: 625) CEP290- +UGCUCUAGAUGACAUGAGGUAAGU 24 downstream 1580 (SEQ ID NO: 2388) CEP290- +UCUAGAUGACAUGAGGUAAGU (SEQ ID 21 downstream 1581 NO: 2389) CEP290- +UAGAUGACAUGAGGUAAGU (SEQ ID 19 downstream 1582 NO: 2390) CEP290- −UAAUACAUGAGAGUGAUUAGUGG 23 downstream 1583 (SEQ ID NO: 2391) CEP290- −UACAUGAGAGUGAUUAGUGG (SEQ ID 20 downstream 1584 NO: 628) CEP290- −UAAAGGUUCAUGAGACUAGAGGUC 24 downstream 1585 (SEQ ID NO: 2392) CEP290- −UUCAUGAGACUAGAGGUC (SEQ ID NO: 18 downstream 1586 2393) CEP290- +UGGCAGUAAGGAGGAUGUAAGAC 23 downstream 1587 (SEQ ID NO: 2394) CEP290- +UAGCUUUUGACAGUUUUUAAGG (SEQ 22 downstream 1588 ID NO: 2395) CEP290- +UUUUGACAGUUUUUAAGG (SEQ ID NO: 18 downstream 1589 2396) CEP290- −UUGUACGUGCUCUUUUCUAUAUAU 24 downstream 1590 (SEQ ID NO: 2397) CEP290- −UGUACGUGCUCUUUUCUAUAUAU (SEQ 23 downstream 1591 ID NO: 2398) CEP290- −UACGUGCUCUUUUCUAUAUAU (SEQ ID 21 downstream 1592 NO: 2399) CEP290- +UUCACUGAGCAAAACAACUGG (SEQ ID 21 downstream 1593 NO: 2400) CEP290- +UCACUGAGCAAAACAACUGG (SEQ ID 20 downstream 1594 NO: 883) CEP290- +UGAACAAGUUUUGAAACAGGAA (SEQ 22 downstream 1595 ID NO: 2402) CEP290- +UAAUGCCUGAACAAGUUUUGAAA 23 downstream 1596 (SEQ ID NO: 2403) CEP290- +UGCCUGAACAAGUUUUGAAA (SEQ ID 20 downstream 1597 NO: 897) CEP290- +UUCACUGAGCAAAACAACUGGAA (SEQ 23 downstream 1598 ID NO: 2405) CEP290- +UCACUGAGCAAAACAACUGGAA (SEQ 22 downstream 1599 ID NO: 2406) CEP290- +UGAGCAAAACAACUGGAA (SEQ ID NO: 18 downstream 1600 2407) CEP290- −UUUGUACGUGCUCUUUUCUAUAUA 24 downstream 1601 (SEQ ID NO: 2408) CEP290- −UUGUACGUGCUCUUUUCUAUAUA (SEQ 23 downstream 1602 ID NO: 2409) CEP290- −UGUACGUGCUCUUUUCUAUAUA (SEQ 22 downstream 1603 ID NO: 2410) CEP290- −UACGUGCUCUUUUCUAUAUA (SEQ ID 20 downstream 1604 NO: 877) CEP290- +UAUUAUCUAUUCCAUUCUUCACAC 24 downstream 1605 (SEQ ID NO: 2412) CEP290- +UUAUCUAUUCCAUUCUUCACAC (SEQ 22 downstream 1606 ID NO: 2413) CEP290- +UAUCUAUUCCAUUCUUCACAC (SEQ ID 21 downstream 1607 NO: 2414) CEP290- +UCUAUUCCAUUCUUCACAC (SEQ ID 19 downstream 1608 NO: 2415) CEP290- −UGCUUAGGAAAUUAUUGUUGCUUU 24 downstream 1609 (SEQ ID NO: 2416) CEP290- −UUAGGAAAUUAUUGUUGCUUU (SEQ 21 downstream 1610 ID NO: 2417) CEP290- −UAGGAAAUUAUUGUUGCUUU (SEQ ID 20 downstream 1611 NO: 2418) CEP290- −UUGCUUUUUGAGAGGUAAAGGUUC 24 downstream 1612 (SEQ ID NO: 2419) CEP290- −UGCUUUUUGAGAGGUAAAGGUUC 23 downstream 1613 (SEQ ID NO: 2420) CEP290- −UUUUUGAGAGGUAAAGGUUC (SEQ ID 20 downstream 1614 NO: 2421) CEP290- −UUUUGAGAGGUAAAGGUUC (SEQ ID 19 downstream 1615 NO: 2422) CEP290- −UUUGAGAGGUAAAGGUUC (SEQ ID NO: 18 downstream 1616 2423) CEP290- +UCACUGAGCAAAACAACUGGAAGA 24 downstream 1617 (SEQ ID NO: 2424) CEP290- +UGAGCAAAACAACUGGAAGA (SEQ ID 20 downstream 1618 NO: 894) CEP290- +UACAUAAGAAAGAACACUGUGGU 23 downstream 1619 (SEQ ID NO: 2426) CEP290- +UAAGAAAGAACACUGUGGU (SEQ ID 19 downstream 1620 NO: 2427) CEP290- −UGUGUGAAGAAUGGAAUAGAUAAU 24 downstream 1621 (SEQ ID NO: 2428) CEP290- −UGUGAAGAAUGGAAUAGAUAAU (SEQ 22 downstream 1622 ID NO: 2429) CEP290- −UGAAGAAUGGAAUAGAUAAU (SEQ ID 20 downstream 1623 NO: 2430) CEP290- +UAAGGAGGAUGUAAGACUGGAGA 23 downstream 1624 (SEQ ID NO: 2431) CEP290- −UGUUUACAUAUCUGUCUUCCUUA (SEQ 23 downstream 1625 ID NO: 2432) CEP290- −UUUACAUAUCUGUCUUCCUUA (SEQ ID 21 downstream 1626 NO: 2433) CEP290- −UUACAUAUCUGUCUUCCUUA (SEQ ID 20 downstream 1627 NO: 901) CEP290- −UACAUAUCUGUCUUCCUUA (SEQ ID 19 downstream 1628 NO: 2435) CEP290- +UGUUCCAUUAAAAAAAGUAUGCUU 24 downstream 1629 (SEQ ID NO: 2436) CEP290- +UUCCAUUAAAAAAAGUAUGCUU (SEQ 22 downstream 1630 ID NO: 2437) CEP290- +UCCAUUAAAAAAAGUAUGCUU (SEQ ID 21 downstream 1631 NO: 2438) CEP290- +UAUCAAAAGACUUAUAUUCCAUU (SEQ 23 downstream 1632 ID NO: 2439) CEP290- +UCAAAAGACUUAUAUUCCAUU (SEQ ID 21 downstream 1633 NO: 2440) CEP290- −UCAGAUUUCAUGUGUGAAGA (SEQ ID 20 downstream 1634 NO: 2441) CEP290- −UGGAAUAUAAGUCUUUUGAUAU (SEQ 22 downstream 1635 ID NO: 2442) CEP290- −UGUGAAGAAUGGAAUAGAUAAUA 23 downstream 1636 (SEQ ID NO: 2443) CEP290- −UGAAGAAUGGAAUAGAUAAUA (SEQ 21 downstream 1637 ID NO: 2444) CEP290- −UGGAUGGGUAAUAAAGCAA (SEQ ID 19 downstream 1638 NO: 2445) CEP290- +UAGAAAUUCACUGAGCAAAACAA (SEQ 23 downstream 1639 ID NO: 2446) CEP290- +UGUAAGACUGGAGAUAGAGA (SEQ ID 20 downstream 1640 NO: 898) CEP290- +UAAGACUGGAGAUAGAGA (SEQ ID NO: 18 downstream 1641 2448) CEP290- −UUGAAAUUUGAUAGUAGAAGAAAA 24 downstream 1642 (SEQ ID NO: 2449) CEP290- −UGAAAUUUGAUAGUAGAAGAAAA 23 downstream 1643 (SEQ ID NO: 2450) CEP290- −UUUGAUAGUAGAAGAAAA (SEQ ID NO: 18 downstream 1644 2451) CEP290- +UAAAACUAAGACACUGCCAA (SEQ ID 20 downstream 1645 NO: 871) CEP290- −UUUUUCUUAAGCAUACUUUUUUUA 24 downstream 1646 (SEQ ID NO: 2453) CEP290- −UUUUCUUAAGCAUACUUUUUUUA 23 downstream 1647 (SEQ ID NO: 2454) CEP290- −UUUCUUAAGCAUACUUUUUUUA (SEQ 22 downstream 1648 ID NO: 2455) CEP290- −UUCUUAAGCAUACUUUUUUUA (SEQ ID 21 downstream 1649 NO: 2456) CEP290- −UCUUAAGCAUACUUUUUUUA (SEQ ID 20 downstream 1650 NO: 891) CEP290- −UUAAGCAUACUUUUUUUA (SEQ ID NO: 18 downstream 1651 2458) CEP290- −UGGGUAAUAAAGCAAAAGAAAAAC 24 downstream 1652 (SEQ ID NO: 2459) CEP290- −UAAUAAAGCAAAAGAAAAAC (SEQ ID 20 downstream 1653 NO: 2460) CEP290- −UUCUUUUUUUGUUGUUUUUUUUU 23 downstream 1654 (SEQ ID NO: 2461) CEP290- −UCUUUUUUUGUUGUUUUUUUUU (SEQ 22 downstream 1655 ID NO: 2462) CEP290- −UUUUUUUGUUGUUUUUUUUU (SEQ ID 20 downstream 1656 NO: 2463) CEP290- −UUUUUUGUUGUUUUUUUUU (SEQ ID 19 downstream 1657 NO: 2464) CEP290- −UUUUUGUUGUUUUUUUUU (SEQ ID NO: 18 downstream 1658 2465) CEP290- +UGCACUCCAGCCUGGGCAACACA (SEQ 23 downstream 1659 ID NO: 2466) CEP290- +UCCAGCCUGGGCAACACA (SEQ ID NO: 18 downstream 1660 2467) CEP290- +AUUUUCGUGACCUCUAGUCUC (SEQ ID 21 downstream 1661 NO: 2468) CEP290- +ACUAAUCACUCUCAUGUAUUAGC (SEQ 23 downstream 1662 ID NO: 2469) CEP290- +AAUCACUCUCAUGUAUUAGC (SEQ ID 20 downstream 1663 NO: 814) CEP290- +AUCACUCUCAUGUAUUAGC (SEQ ID 19 downstream 1664 NO: 2471) CEP290- +AGAUGACAUGAGGUAAGUA (SEQ ID 19 downstream 1665 NO: 2472) CEP290- −ACCUCAUGUCAUCUAGAGCAAGAG 24 downstream 1666 (SEQ ID NO: 2473) CEP290- −AUGUCAUCUAGAGCAAGAG (SEQ ID 19 downstream 1667 NO: 2474) CEP290- −AAUACAUGAGAGUGAUUAGUGGUG 24 downstream 1668 (SEQ ID NO: 2475) CEP290- −AUACAUGAGAGUGAUUAGUGGUG 23 downstream 1669 (SEQ ID NO: 2476) CEP290- −ACAUGAGAGUGAUUAGUGGUG (SEQ 21 downstream 1670 ID NO: 2477) CEP290- −AUGAGAGUGAUUAGUGGUG (SEQ ID 19 downstream 1671 NO: 2478) CEP290- −ACGUGCUCUUUUCUAUAUAUA (SEQ ID 21 downstream 1672 NO: 2479) CEP290- +ACAAAACCUAUGUAUAAGAUG (SEQ ID 21 downstream 1673 NO: 2480) CEP290- +AAAACCUAUGUAUAAGAUG (SEQ ID 19 downstream 1674 NO: 2481) CEP290- +AAACCUAUGUAUAAGAUG (SEQ ID NO: 18 downstream 1675 2482) CEP290- +AUAUAUAGAAAAGAGCACGUACAA 24 downstream 1676 (SEQ ID NO: 2483) CEP290- +AUAUAGAAAAGAGCACGUACAA (SEQ 22 downstream 1677 ID NO: 2484) CEP290- +AUAGAAAAGAGCACGUACAA (SEQ ID 20 downstream 1678 NO: 832) CEP290- +AGAAAAGAGCACGUACAA (SEQ ID NO: 18 downstream 1679 2486) CEP290- +AGAAAUGGUUCCCUAUAUAUAGAA 24 downstream 1680 (SEQ ID NO: 2487) CEP290- +AAAUGGUUCCCUAUAUAUAGAA (SEQ 22 downstream 1681 ID NO: 2488) CEP290- +AAUGGUUCCCUAUAUAUAGAA (SEQ ID 21 downstream 1682 NO: 2489) CEP290- +AUGGUUCCCUAUAUAUAGAA (SEQ ID 20 downstream 1683 NO: 839) CEP290- −AUGGAAUAUAAGUCUUUUGAUAUA 24 downstream 1684 (SEQ ID NO: 2491) CEP290- −AAUAUAAGUCUUUUGAUAUA (SEQ ID 20 downstream 1685 NO: 687) CEP290- −AUAUAAGUCUUUUGAUAUA (SEQ ID 19 downstream 1686 NO: 2493) CEP290- +ACGUACAAAAGAACAUACAUAAGA 24 downstream 1687 (SEQ ID NO: 2494) CEP290- +ACAAAAGAACAUACAUAAGA (SEQ ID 20 downstream 1688 NO: 816) CEP290- +AAAAGAACAUACAUAAGA (SEQ ID NO: 18 downstream 1689 2496) CEP290- +AAGAAAAAAAAGGUAAUGC (SEQ ID 19 downstream 1690 NO: 2497) CEP290- +AGAAAAAAAAGGUAAUGC (SEQ ID NO: 18 downstream 1691 2498) CEP290- +AAACAGGAAUAGAAAUUCA (SEQ ID 19 downstream 1692 NO: 2499) CEP290- +AACAGGAAUAGAAAUUCA (SEQ ID NO: 18 downstream 1693 2500) CEP290- +AAGAUCACUCCACUGCACUCCAGC 24 downstream 1694 (SEQ ID NO: 2501) CEP290- +AGAUCACUCCACUGCACUCCAGC (SEQ 23 downstream 1695 ID NO: 2502) CEP290- +AUCACUCCACUGCACUCCAGC (SEQ ID 21 downstream 1696 NO: 2503) CEP290- +ACUCCACUGCACUCCAGC (SEQ ID NO: 18 downstream 1697 2504) CEP290- −CCCCUACUUACCUCAUGUCAUC (SEQ 22 downstream 1698 ID NO: 2505) CEP290- −CCCUACUUACCUCAUGUCAUC (SEQ ID 21 downstream 1699 NO: 2506) CEP290- −CCUACUUACCUCAUGUCAUC (SEQ ID 20 downstream 1700 NO: 747) CEP290- −CUACUUACCUCAUGUCAUC (SEQ ID 19 downstream 1701 NO: 2508) CEP290- +CUGAUUUUCGUGACCUCUAGUCUC 24 downstream 1702 (SEQ ID NO: 2509) CEP290- +CACUAAUCACUCUCAUGUAUUAGC 24 downstream 1703 (SEQ ID NO: 2510) CEP290- +CUAAUCACUCUCAUGUAUUAGC (SEQ 22 downstream 1704 ID NO: 2511) CEP290- +CUCUAGAUGACAUGAGGUAAGUA 23 downstream 1705 (SEQ ID NO: 2512) CEP290- +CUAGAUGACAUGAGGUAAGUA (SEQ ID 21 downstream 1706 NO: 2513) CEP290- −CCUCAUGUCAUCUAGAGCAAGAG (SEQ 23 downstream 1707 ID NO: 2514) CEP290- −CUCAUGUCAUCUAGAGCAAGAG (SEQ 22 downstream 1708 ID NO: 2515) CEP290- −CAUGUCACUAGAGCAAGAG (SEQ ID 20 downstream 1709 NO: 855) CEP290- −CAUGAGAGUGAUUAGUGGUG (SEQ ID 20 downstream 1710 NO: 854) CEP290- −CGUGCUCUUUUCUAUAUAUA (SEQ ID 20 downstream 1711 NO: 624) CEP290- +CAAAACCUAUGUAUAAGAUG (SEQ ID 20 downstream 1712 NO: 841) CEP290- +CGUACAAAAGAACAUACAUAAGA (SEQ 23 downstream 1713 ID NO: 2520) CEP290- +CAAAAGAACAUACAUAAGA (SEQ ID 19 downstream 1714 NO: 2521) CEP290- +CUUAAGAAAAAAAAGGUAAUGC (SEQ 22 downstream 1715 ID NO: 2522) CEP290- −CUUAAGCAUACUUUUUUUAA (SEQ ID 20 downstream 1716 NO: 690) CEP290- +CACUCCACUGCACUCCAGC (SEQ ID 19 downstream 1717 NO: 2524) CEP290-132 −GUCCCCUACUUACCUCAUGUCAUC 24 downstream (SEQ ID NO: 2525) CEP290- +GAUUUUCGUGACCUCUAGUCUC (SEQ 22 downstream 1718 ID NO: 2526) CEP290- +GCUCUAGAUGACAUGAGGUAAGUA 24 downstream 1719 (SEQ ID NO: 2527) CEP290- +GAUGACAUGAGGUAAGUA (SEQ ID NO: 18 downstream 1720 2528) CEP290- −GUACGUGCUCUUUUCUAUAUAUA (SEQ 23 downstream 1721 ID NO: 2529) CEP290- −GUGCUCUUUUCUAUAUAUA (SEQ ID 19 downstream 1722 NO: 2530) CEP290- +GUACAAAACCUAUGUAUAAGAUG 23 downstream 1723 (SEQ ID NO: 2531) CEP290- +GAAAUGGUUCCCUAUAUAUAGAA 23 downstream 1724 (SEQ ID NO: 2532) CEP290- +GGUUCCCUAUAUAUAGAA (SEQ ID NO: 18 downstream 1725 2533) CEP290- −GGAAUAUAAGUCUUUUGAUAUA (SEQ 22 downstream 1726 ID NO: 2534) CEP290- −GAAUAUAAGUCUUUUGAUAUA (SEQ 21 downstream 1727 ID NO: 2535) CEP290- +GUACAAAAGAACAUACAUAAGA (SEQ 22 downstream 1728 ID NO: 2536) CEP290- +GCUUAAGAAAAAAAAGGUAAUGC 23 downstream 1729 (SEQ ID NO: 2537) CEP290- +GAAACAGGAAUAGAAAUUCA (SEQ ID 20 downstream 1730 NO: 769) CEP290- +GAUCACUCCACUGCACUCCAGC (SEQ 22 downstream 1731 ID NO: 2539) CEP290- −UCCCCUACUUACCUCAUGUCAUC (SEQ 23 downstream 1732 ID NO: 2540) CEP290- −UACUUACCUCAUGUCAUC (SEQ ID NO: 18 downstream 1733 2541) CEP290- +UGAUUUUCGUGACCUCUAGUCUC (SEQ 23 downstream 1734 ID NO: 2542) CEP290- +UUUUCGUGACCUCUAGUCUC (SEQ ID 20 downstream 1735 NO: 2543) CEP290- +UUUCGUGACCUCUAGUCUC (SEQ ID 19 downstream 1736 NO: 2544) CEP290- +UUCGUGACCUCUAGUCUC (SEQ ID NO: 18 downstream 1737 2545) CEP290- +UAAUCACUCUCAUGUAUUAGC (SEQ ID 21 downstream 1738 NO: 2546) CEP290- +UCACUCUCAUGUAUUAGC (SEQ ID NO: 18 downstream 1739 2547) CEP290- +UCUAGAUGACAUGAGGUAAGUA (SEQ 22 downstream 1740 ID NO: 2548) CEP290- +UAGAUGACAUGAGGUAAGUA (SEQ ID 20 downstream 1741 NO: 680) CEP290- −UCAUGUCAUCUAGAGCAAGAG (SEQ ID 21 downstream 1742 NO: 2550) CEP290- −UGUCAUCUAGAGCAAGAG (SEQ ID NO: 18 downstream 1743 2551) CEP290- −UACAUGAGAGUGAUUAGUGGUG (SEQ 22 downstream 1744 ID NO: 2552) CEP290- −UGAGAGUGAUUAGUGGUG (SEQ ID NO: 18 downstream 1745 2553) CEP290- −UGUACGUGCUCUUUUCUAUAUAUA 24 downstream 1746 (SEQ ID NO: 2554) CEP290- −UACGUGCUCUUUUCUAUAUAUA (SEQ 22 downstream 1747 ID NO: 2555) CEP290- −UGCUCUUUUCUAUAUAUA (SEQ ID NO: 18 downstream 1748 2556) CEP290- +UGUACAAAACCUAUGUAUAAGAUG 24 downstream 1749 (SEQ ID NO: 2557) CEP290- +UACAAAACCUAUGUAUAAGAUG (SEQ 22 downstream 1750 ID NO: 2558) CEP290- +UAUAUAGAAAAGAGCACGUACAA 23 downstream 1751 (SEQ ID NO: 2559) CEP290- +UAUAGAAAAGAGCACGUACAA (SEQ ID 21 downstream 1752 NO: 2560) CEP290- +UAGAAAAGAGCACGUACAA (SEQ ID 19 downstream 1753 NO: 2561) CEP290- +UGGUUCCCUAUAUAUAGAA (SEQ ID 19 downstream 1754 NO: 2562) CEP290- −UGGAAUAUAAGUCUUUUGAUAUA 23 downstream 1755 (SEQ ID NO: 2563) CEP290- −UAUAAGUCUUUUGAUAUA (SEQ ID NO: 18 downstream 1756 2564) CEP290- +UACAAAAGAACAUACAUAAGA (SEQ ID 21 downstream 1757 NO: 2565) CEP290- +UGCUUAAGAAAAAAAAGGUAAUGC 24 downstream 1758 (SEQ ID NO: 2566) CEP290- +UUAAGAAAAAAAAGGUAAUGC (SEQ 21 downstream 1759 ID NO: 2567) CEP290- +UAAGAAAAAAAAGGUAAUGC (SEQ ID 20 downstream 1760 NO: 872) CEP290- +UUUUGAAACAGGAAUAGAAAUUCA 24 downstream 1761 (SEQ ID NO: 2569) CEP290- +UUUGAAACAGGAAUAGAAAUUCA 23 downstream 1762 (SEQ ID NO: 2570) CEP290- +UUGAAACAGGAAUAGAAAUUCA (SEQ 22 downstream 1763 ID NO: 2571) CEP290- +UGAAACAGGAAUAGAAAUUCA (SEQ ID 21 downstream 1764 NO: 2572) CEP290- −UUUUCUUAAGCAUACUUUUUUUAA 24 downstream 1765 (SEQ ID NO: 2573) CEP290- −UUUCUUAAGCAUACUUUUUUUAA 23 downstream 1766 (SEQ ID NO: 2574) CEP290- −UUCUUAAGCAUACUUUUUUUAA (SEQ 22 downstream 1767 ID NO: 2575) CEP290- −UCUUAAGCAUACUUUUUUUAA (SEQ ID 21 downstream 1768 NO: 2576) CEP290- −UUAAGCAUACUUUUUUUAA (SEQ ID 19 downstream 1769 NO: 2577) CEP290- −UAAGCAUACUUUUUUUAA (SEQ ID NO: 18 downstream 1770 2578) CEP290- +UCACUCCACUGCACUCCAGC (SEQ ID 20 downstream 1771 NO: 2579) CEP290- +AGUUUUUAAGGCGGGGAGUCACA 23 downstream 1772 (SEQ ID NO: 2580) CEP290- −AAACUGUCAAAAGCUACCGGUUAC 24 downstream 1773 (SEQ ID NO: 2581) CEP290- −AACUGUCAAAAGCUACCGGUUAC (SEQ 23 downstream 1774 ID NO: 2582) CEP290-252− ACUGUCAAAAGCUACCGGUUAC (SEQ 22 downstream ID NO: 2583) CEP290- +AGUUCAUCUCUUGCUCUAGAUGAC 24 downstream 1775 (SEQ ID NO: 2584) CEP290- +AUCUCUUGCUCUAGAUGAC (SEQ ID 19 downstream 1776 NO: 2585) CEP290- −ACGAAAAUCAGAUUUCAUGU (SEQ ID 20 downstream 1777 NO: 2586) CEP290- −AAUACAUGAGAGUGAUUAGUG (SEQ 21 downstream 1778 ID NO: 2587) CEP290- −AUACAUGAGAGUGAUUAGUG (SEQ ID 20 downstream 1779 NO: 831) CEP290- −ACAUGAGAGUGAUUAGUG (SEQ ID NO: 18 downstream 1780 2589) CEP290- +AUUAGCUUGAACUCUGUGCCAAA (SEQ 23 downstream 1781 ID NO: 2590) CEP290- +AGCUUGAACUCUGUGCCAAA (SEQ ID 20 downstream 1782 NO: 824) CEP290- −AUGUAGAUUGAGGUAGAAUCAAG 23 downstream 1783 (SEQ ID NO: 2592) CEP290- −AGAUUGAGGUAGAAUCAAG (SEQ ID 19 downstream 1784 NO: 2593) CEP290- +AUAAGAUGCAGAACUAGUGUAGA 23 downstream 1785 (SEQ ID NO: 2594) CEP290- +AAGAUGCAGAACUAGUGUAGA (SEQ ID 21 downstream 1786 NO: 2595) CEP290- +AGAUGCAGAACUAGUGUAGA (SEQ ID 20 downstream 1787 NO: 821) CEP290- +AUGCAGAACUAGUGUAGA (SEQ ID NO: 18 downstream 1788 2597) CEP290- −AUAGAUGUAGAUUGAGGUAGAAUC 24 downstream 1789 (SEQ ID NO: 2598) CEP290- −AGAUGUAGAUUGAGGUAGAAUC (SEQ 22 downstream 1790 ID NO: 2599) CEP290- −AUGUAGAUUGAGGUAGAAUC (SEQ ID 20 downstream 1791 NO: 2600) CEP290- +AGAAUGAUCAUUCUUGUGGCAGUA 24 downstream 1792 (SEQ ID NO: 2601) CEP290- +AAUGAUCAUUCUUGUGGCAGUA (SEQ 22 downstream 1793 ID NO: 2602) CEP290- +AUGAUCAUUCUUGUGGCAGUA (SEQ ID 21 downstream 1794 NO: 2603) CEP290- +AUCAUUCUUGUGGCAGUA (SEQ ID NO: 18 downstream 1795 2604) CEP290- +AGAAUGAUCAUUCUUGUGGCAGU 23 downstream 1796 (SEQ ID NO: 2605) CEP290- +AAUGAUCAUUCUUGUGGCAGU (SEQ ID 21 downstream 1797 NO: 2606) CEP290- +AUGAUCAUUCUUGUGGCAGU (SEQ ID 20 downstream 1798 NO: 837) CEP290- −AGAGGUAAAGGUUCAUGAGAC (SEQ ID 21 downstream 1799 NO: 2608) CEP290- −AGGUAAAGGUUCAUGAGAC (SEQ ID 19 downstream 1800 NO: 2609) CEP290- +AGCUUUUGACAGUUUUUAAG (SEQ ID 20 downstream 1801 NO: 825) CEP290- +AGCUUUUGACAGUUUUUAAGGC (SEQ 22 downstream 1802 ID NO: 2611) CEP290- +AGAAAUUCACUGAGCAAAACAAC (SEQ 23 downstream 1803 ID NO: 2612) CEP290- +AAAUUCACUGAGCAAAACAAC (SEQ ID 21 downstream 1804 NO: 2613) CEP290- +AAUUCACUGAGCAAAACAAC (SEQ ID 20 downstream 1805 NO: 678) CEP290- +AUUCACUGAGCAAAACAAC (SEQ ID 19 downstream 1806 NO: 2615) CEP290- +AGUAAGGAGGAUGUAAGA (SEQ ID NO: 18 downstream 1807 2616) CEP290- +AUCAAAAGACUUAUAUUCCAUUA (SEQ 23 downstream 1808 ID NO: 2617) CEP290- +AAAAGACUUAUAUUCCAUUA (SEQ ID 20 downstream 1809 NO: 685) CEP290- +AAAGACUUAUAUUCCAUUA (SEQ ID 19 downstream 1810 NO: 2619) CEP290- +AAGACUUAUAUUCCAUUA (SEQ ID NO: 18 downstream 1811 2620) CEP290- −AGGAAAUUAUUGUUGCUUUUU (SEQ 21 downstream 1812 ID NO: 2621) CEP290- −AAAUUAUUGUUGCUUUUU (SEQ ID NO: 18 downstream 1813 2622) CEP290- −AAAGAAAAACUUGAAAUUUGAUAG 24 downstream 1814 (SEQ ID NO: 2623) CEP290- −AAGAAAAACUUGAAAUUUGAUAG 23 downstream 1815 (SEQ ID NO: 2624) CEP290- −AGAAAAACUUGAAAUUUGAUAG (SEQ 22 downstream 1816 ID NO: 2625) CEP290- −AAAAACUUGAAAUUUGAUAG (SEQ ID 20 downstream 1817 NO: 2626) CEP290- −AAAACUUGAAAUUUGAUAG (SEQ ID 19 downstream 1818 NO: 2627) CEP290- −AAACUUGAAAUUUGAUAG (SEQ ID NO: 18 downstream 1819 2628) CEP290- −AAGAAAAAAGAAAUAGAUGUAGA 23 downstream 1820 (SEQ ID NO: 2629) CEP290- −AGAAAAAAGAAAUAGAUGUAGA (SEQ 22 downstream 1821 ID NO: 2630) CEP290- −AAAAAAGAAAUAGAUGUAGA (SEQ ID 20 downstream 1822 NO: 2631) CEP290- −AAAAAGAAAUAGAUGUAGA (SEQ ID 19 downstream 1823 NO: 2632) CEP290- −AAAAGAAAUAGAUGUAGA (SEQ ID NO: 18 downstream 1824 2633) CEP290- −AGAGUCUCACUGUGUUGCCCAGG (SEQ 23 downstream 1825 ID NO: 2634) CEP290- −AGUCUCACUGUGUUGCCCAGG (SEQ ID 21 downstream 1826 NO: 2635) CEP290- +CAGUUUUUAAGGCGGGGAGUCACA 24 downstream 1827 (SEQ ID NO: 2636) CEP290- −CUGUCAAAAGCUACCGGUUAC (SEQ ID 21 downstream 1828 NO: 2637) CEP290- +CAUCUCUUGCUCUAGAUGAC (SEQ ID 20 downstream 1829 NO: 853) CEP290- −CACGAAAAUCAGAUUUCAUGU (SEQ ID 21 downstream 1830 NO: 2639) CEP290- −CGAAAAUCAGAUUUCAUGU (SEQ ID 19 downstream 1831 NO: 2640) CEP290- −CUAAUACAUGAGAGUGAUUAGUG 23 downstream 1832 (SEQ ID NO: 2641) CEP290- +CUUGAACUCUGUGCCAAA (SEQ ID NO: 18 downstream 1833 2642) CEP290- +CUCUAGAUGACAUGAGGUAAG (SEQ ID 21 downstream 1834 NO: 2643) CEP290- +CUAGAUGACAUGAGGUAAG (SEQ ID 19 downstream 1835 NO: 2644) CEP290- +CGGUAGCUUUUGACAGUUUUUAAG 24 downstream 1836 (SEQ ID NO: 2645) CEP290- +CUUUUGACAGUUUUUAAG (SEQ ID NO: 18 downstream 1837 2646) CEP290- +CUUUUGACAGUUUUUAAGGC (SEQ ID 20 downstream 1838 NO: 684) CEP290- +CAGUAAGGAGGAUGUAAGA (SEQ ID 19 downstream 1839 NO: 2648) CEP290- +CAAAAGACUUAUAUUCCAUUA (SEQ ID 21 downstream 1840 NO: 2649) CEP290- −CUUAGGAAAUUAUUGUUGCUUUUU 24 downstream 1841 (SEQ ID NO: 2650) CEP290- −CUGUGUUGCCCAGGCUGGAGUGCA 24 downstream 1842 (SEQ ID NO: 2651) CEP290- −CAGAGUCUCACUGUGUUGCCCAGG 24 downstream 1843 (SEQ ID NO: 2652) CEP290- −CUCACUGUGUUGCCCAGG (SEQ ID NO: 18 downstream 1844 2653) CEP290- +GUUUUUAAGGCGGGGAGUCACA (SEQ 22 downstream 1845 ID NO: 2654) CEP290- −GUCAAAAGCUACCGGUUAC (SEQ ID 19 downstream 1846 NO: 2655) CEP290- +GUUCAUCUCUUGCUCUAGAUGAC (SEQ 23 downstream 1847 ID NO: 2656) CEP290- −GGUCACGAAAAUCAGAUUUCAUGU 24 downstream 1848 (SEQ ID NO: 2657) CEP290- −GUCACGAAAAUCAGAUUUCAUGU (SEQ 23 downstream 1849 ID NO: 2658) CEP290- −GAAAAUCAGAUUUCAUGU (SEQ ID NO: 18 downstream 1850 2659) CEP290- −GCUAAUACAUGAGAGUGAUUAGUG 24 downstream 1851 (SEQ ID NO: 2660) CEP290- +GCUUGAACUCUGUGCCAAA (SEQ ID 19 downstream 1852 NO: 2661) CEP290- +GCUCUAGAUGACAUGAGGUAAG (SEQ 22 downstream 1853 ID NO: 2662) CEP290- −GAUGUAGAUUGAGGUAGAAUCAAG 24 downstream 1854 (SEQ ID NO: 2663) CEP290- −GUAGAUUGAGGUAGAAUCAAG (SEQ 21 downstream 1855 ID NO: 2664) CEP290- −GAUUGAGGUAGAAUCAAG (SEQ ID NO: 18 downstream 1856 2665) CEP290- +GAUGCAGAACUAGUGUAGA (SEQ ID 19 downstream 1857 NO: 2666) CEP290- −GAUGUAGAUUGAGGUAGAAUC (SEQ 21 downstream 1858 ID NO: 2667) CEP290- −GUAGAUUGAGGUAGAAUC (SEQ ID NO: 18 downstream 1859 2668) CEP290- +GAAUGAUCAUUCUUGUGGCAGUA 23 downstream 1860 (SEQ ID NO: 2669) CEP290- +GAUCAUUCUUGUGGCAGUA (SEQ ID 19 downstream 1861 NO: 2670) CEP290- +GAAUGAUCAUUCUUGUGGCAGU (SEQ 22 downstream 1862 ID NO: 2671) CEP290- +GAUCAUUCUUGUGGCAGU (SEQ ID NO: 18 downstream 1863 2672) CEP290- −GAGAGGUAAAGGUUCAUGAGAC (SEQ 22 downstream 1864 ID NO: 2673) CEP290- −GAGGUAAAGGUUCAUGAGAC (SEQ ID 20 downstream 1865 NO: 2674) CEP290- −GGUAAAGGUUCAUGAGAC (SEQ ID NO: 18 downstream 1866 2675) CEP290- +GGUAGCUUUUGACAGUUUUUAAG 23 downstream 1867 (SEQ ID NO: 2676) CEP290- +GUAGCUUUUGACAGUUUUUAAG (SEQ 22 downstream 1868 ID NO: 2677) CEP290- +GCUUUUGACAGUUUUUAAG (SEQ ID 19 downstream 1869 NO: 2678) CEP290- +GUAGCUUUUGACAGUUUUUAAGGC 24 downstream 1870 (SEQ ID NO: 2679) CEP290- +GCUUUUGACAGUUUUUAAGGC (SEQ ID 21 downstream 1871 NO: 2680) CEP290- +GAAAUUCACUGAGCAAAACAAC (SEQ 22 downstream 1872 ID NO: 2681) CEP290- +GUGGCAGUAAGGAGGAUGUAAGA 23 downstream 1873 (SEQ ID NO: 2682) CEP290- +GGCAGUAAGGAGGAUGUAAGA (SEQ 21 downstream 1874 ID NO: 2683) CEP290- +GCAGUAAGGAGGAUGUAAGA (SEQ ID 20 downstream 1875 NO: 775) CEP290- −GGAAAUUAUUGUUGCUUUUU (SEQ ID 20 downstream 1876 NO: 2685) CEP290- −GAAAUUAUUGUUGCUUUUU (SEQ ID 19 downstream 1877 NO: 2686) CEP290- −GAAAAACUUGAAAUUUGAUAG (SEQ 21 downstream 1878 ID NO: 2687) CEP290- −GAAGAAAAAAGAAAUAGAUGUAGA 24 downstream 1879 (SEQ ID NO: 2688) CEP290- −GAAAAAAGAAAUAGAUGUAGA (SEQ 21 downstream 1880 ID NO: 2689) CEP290- −GUGUUGCCCAGGCUGGAGUGCA (SEQ 22 downstream 1881 ID NO: 2690) CEP290- −GUUGCCCAGGCUGGAGUGCA (SEQ ID 20 downstream 1882 NO: 2691) CEP290- −GAGUCUCACUGUGUUGCCCAGG (SEQ 22 downstream 1883 ID NO: 2692) CEP290- −GUCUCACUGUGUUGCCCAGG (SEQ ID 20 downstream 1884 NO: 2693) CEP290- +UUUUUAAGGCGGGGAGUCACA (SEQ ID 21 downstream 1885 NO: 2694) CEP290- +UUUUAAGGCGGGGAGUCACA (SEQ ID 20 downstream 1886 NO: 672) CEP290- +UUUAAGGCGGGGAGUCACA (SEQ ID 19 downstream 1887 NO: 2696) CEP290- +UUAAGGCGGGGAGUCACA (SEQ ID NO: 18 downstream 1888 2697) CEP290- −UGUCAAAAGCUACCGGUUAC (SEQ ID 20 downstream 1889 NO: 757) CEP290- −UCAAAAGCUACCGGUUAC (SEQ ID NO: 18 downstream 1890 2699) CEP290- +UUCAUCUCUUGCUCUAGAUGAC (SEQ 22 downstream 1891 ID NO: 2700) CEP290- +UCAUCUCUUGCUCUAGAUGAC (SEQ ID 21 downstream 1892 NO: 2701) CEP290- +UCUCUUGCUCUAGAUGAC (SEQ ID NO: 18 downstream 1893 2702) CEP290- −UCACGAAAAUCAGAUUUCAUGU (SEQ 22 downstream 1894 ID NO: 2703) CEP290- −UAAUACAUGAGAGUGAUUAGUG (SEQ 22 downstream 1895 ID NO: 2704) CEP290- −UACAUGAGAGUGAUUAGUG (SEQ ID 19 downstream 1896 NO: 2705) CEP290- +UAUUAGCUUGAACUCUGUGCCAAA 24 downstream 1897 (SEQ ID NO: 2706) CEP290- +UUAGCUUGAACUCUGUGCCAAA (SEQ 22 downstream 1898 ID NO: 2707) CEP290- +UAGCUUGAACUCUGUGCCAAA (SEQ ID 21 downstream 1899 NO: 2708) CEP290- +UUGCUCUAGAUGACAUGAGGUAAG 24 downstream 1900 (SEQ ID NO: 2709) CEP290- +UGCUCUAGAUGACAUGAGGUAAG 23 downstream 1901 (SEQ ID NO: 2710) CEP290- +UCUAGAUGACAUGAGGUAAG (SEQ ID 20 downstream 1902 NO: 888) CEP290- +UAGAUGACAUGAGGUAAG (SEQ ID NO: 18 downstream 1903 2712) CEP290- −UGUAGAUUGAGGUAGAAUCAAG (SEQ 22 downstream 1904 ID NO: 2713) CEP290- −UAGAUUGAGGUAGAAUCAAG (SEQ ID 20 downstream 1905 NO: 2714) CEP290- +UAUAAGAUGCAGAACUAGUGUAGA 24 downstream 1906 (SEQ ID NO: 2715) CEP290- +UAAGAUGCAGAACUAGUGUAGA (SEQ 22 downstream 1907 ID NO: 2716) CEP290- −UAGAUGUAGAUUGAGGUAGAAUC 23 downstream 1908 (SEQ ID NO: 2717) CEP290- −UGUAGAUUGAGGUAGAAUC (SEQ ID 19 downstream 1909 NO: 2718) CEP290- +UGAUCAUUCUUGUGGCAGUA (SEQ ID 20 downstream 1910 NO: 688) CEP290- +UAGAAUGAUCAUUCUUGUGGCAGU 24 downstream 1911 (SEQ ID NO: 2720) CEP290- +UGAUCAUUCUUGUGGCAGU (SEQ ID 19 downstream 1912 NO: 2721) CEP290- −UUGAGAGGUAAAGGUUCAUGAGAC 24 downstream 1913 (SEQ ID NO: 2722) CEP290- −UGAGAGGUAAAGGUUCAUGAGAC 23 downstream 1914 (SEQ ID NO: 2723) CEP290- +UAGCUUUUGACAGUUUUUAAG (SEQ ID 21 downstream 1915 NO: 2724) CEP290- +UAGCUUUUGACAGUUUUUAAGGC 23 downstream 1916 (SEQ ID NO: 2725) CEP290- +UUUUGACAGUUUUUAAGGC (SEQ ID 19 downstream 1917 NO: 2726) CEP290- +UUUGACAGUUUUUAAGGC (SEQ ID NO: 18 downstream 1918 2727) CEP290- +UAGAAAUUCACUGAGCAAAACAAC 24 downstream 1919 (SEQ ID NO: 2728) CEP290- +UUCACUGAGCAAAACAAC (SEQ ID NO: 18 downstream 1920 2729) CEP290- +UGUGGCAGUAAGGAGGAUGUAAGA 24 downstream 1921 (SEQ ID NO: 2730) CEP290- +UGGCAGUAAGGAGGAUGUAAGA (SEQ 22 downstream 1922 ID NO: 2731) CEP290- +UAUCAAAAGACUUAUAUUCCAUUA 24 downstream 1923 (SEQ ID NO: 2732) CEP290- +UCAAAAGACUUAUAUUCCAUUA (SEQ 22 downstream 1924 ID NO: 2733) CEP290- −UUAGGAAAUUAUUGUUGCUUUUU 23 downstream 1925 (SEQ ID NO: 2734) CEP290- −UAGGAAAUUAUUGUUGCUUUUU (SEQ 22 downstream 1926 ID NO: 2735) CEP290- −UGUGUUGCCCAGGCUGGAGUGCA (SEQ 23 downstream 1927 ID NO: 2736) CEP290- −UGUUGCCCAGGCUGGAGUGCA (SEQ ID 21 downstream 1928 NO: 2737) CEP290- −UUGCCCAGGCUGGAGUGCA (SEQ ID 19 downstream 1929 NO: 2738) CEP290- −UGCCCAGGCUGGAGUGCA (SEQ ID NO: 18 downstream 1930 2739) CEP290- −UCUCACUGUGUUGCCCAGG (SEQ ID 19 downstream 1931 NO: 2740) CEP290-13 +AUGAGAUACUCACAAUUACAAC (SEQ 22 upstream ID NO: 1049) CEP290-18 +GUAUGAGAUACUCACAAUUACAAC 24 upstream (SEQ ID NO: 1051) CEP290-14 +UAUGAGAUACUCACAAUUACAAC (SEQ 23 upstream ID NO: 1053) CEP290-19 +GGUAUGAGAUAUUCACAAUUACAA 24 upstream (SEQ ID NO: 1057)

Table 10A provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the first tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation, havegood orthogonality, and start with G. It is contemplated herein that thetargeting domain hybridizes to the target domain through complementarybase pairing. Any of the targeting domains in the table can be used witha N. meningitidis Cas9 molecule that generates a double stranded break(Cas9 nuclease) or a single-stranded break (Cas9 nickase).

TABLE 10A Target Position DNA Targeting Site relative to gRNA NameStrand Domain Length mutation CEP290-1932 + GGCAAAAGCAGCAG 20 upstreamAAAGCA (SEQ ID NO: 591) CEP290-1933 − GUGGCUGAAUGACU 17 upstream UCU(SEQ ID NO: 592) CEP290-1934 − GUUGUUCUGAGUAG 17 upstream CUU (SEQ IDNO: 590) CEP290-1935 − GACUAGAGGUCACG 17 downstream AAA (SEQ ID NO: 593)CEP290-1936 − GAGUUCAAGCUAAU 20 downstream ACAUGA (SEQ ID NO: 589)

Table 10B provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene selected according to the second tier parameters. Thetargeting domains are within 1000 bp upstream of an Alu repeat, within40 bp upstream of mutation, or 1000 bp downstream of the mutation, havegood orthogonality, and do not start with G. It is contemplated hereinthat the targeting domain hybridizes to the target domain throughcomplementary base pairing. Any of the targeting domains in the tablecan be used with a N. meningitidis Cas9 molecule that generates a doublestranded break (Cas9 nuclease) or a single-stranded break (Cas9nickase).

TABLE 10B Target Position DNA Site relative to gRNA Name StrandTargeting Domain Length mutation CEP290-1937 + AAAAGCAGCAGAAAGCA 17upstream (SEQ ID NO: 1012) CEP290-1938 − AACGUUGUUCUGAGUAGCUU 20upstream (SEQ ID NO: 1014) CEP290-1939 − AAUAGAGGCUUAUGGAU 17 upstream(SEQ ID NO: 1007) CEP290-1940 + ACUUAAUGAGUGCUUCCCUC 20 upstream (SEQ IDNO: 2748) CEP290-1941 − AGAAAUAGAGGCUUAUGGA 20 upstream U (SEQ ID NO:1016) CEP290-1942 + AGCAGAAAGCAAACUGA 17 upstream (SEQ ID NO: 1011)CEP290-1943 + AGCAGCAGAAAGCAAACUGA 20 upstream (SEQ ID NO: 1018)CEP290-1944 + AGGGUCUGGUCCAUAUU 17 upstream (SEQ ID NO: 2752)CEP290-1945 − AUAGUGGCUGAAUGACUUCU 20 upstream (SEQ ID NO: 2753)CEP290-1946 + AUGUCUGGUUAAAAGAG 17 upstream (SEQ ID NO: 2754)CEP290-1947 + CAAAGGGUCUGGUCCAUAUU 20 upstream (SEQ ID NO: 2755)CEP290-1948 − CAUCAGAAAUAGAGGCU 17 upstream (SEQ ID NO: 1009)CEP290-1949 − CCUCAUCAGAAAUAGAGGCU 20 upstream (SEQ ID NO: 1017)CEP290-1950 − CUGAGGACAGAACAAGC 17 upstream (SEQ ID NO: 1008)CEP290-1951 − CUGCUGCUUUUGCCAAAGAG 20 upstream (SEQ ID NO: 725)CEP290-1952 − CUGCUUUUGCCAAAGAG 17 upstream (SEQ ID NO: 711)CEP290-1953 + UAAUGAGUGCUUCCCUC 17 upstream (SEQ ID NO: 2761)CEP290-1954 + UAGAUGUCUGGUUAAAAGA 20 upstream G (SEQ ID NO: 2762)CEP290-1955 − UCAUUCUCCUUAGGUCACUU 20 upstream (SEQ ID NO: 2763)CEP290-1956 − UUACUGAGGACAGAACAAGC 20 upstream (SEQ ID NO: 1013)CEP290-1957 − UUCUCCUUAGGUCACUU 17 upstream (SEQ ID NO: 2765)CEP290-1958 − AAGAAAAAAGAAAUAGA 17 downstream (SEQ ID NO: 2766)CEP290-1959 − AGAUUGAGGUAGAAUCAAG 20 downstream A (SEQ ID NO: 2767)CEP290-1960 + AGUCACAUGGGAGUCACAGG 20 downstream (SEQ ID NO: 1006)CEP290-1961 + CAAAAAAAGAAUCCUCU 17 downstream (SEQ ID NO: 2769)CEP290-1962 + CAACAAAAAAAGAAUCCUCU 20 downstream (SEQ ID NO: 2770)CEP290-1963 + CACAUGGGAGUCACAGG 17 downstream (SEQ ID NO: 1005)CEP290-1964 + CAUUCUUCACACAUGAA 17 downstream (SEQ ID NO: 2772)CEP290-1965 − UAGAAGAAAAAAGAAAUAG 20 downstream A (SEQ ID NO: 2773)CEP290-1966 − UGAGACUAGAGGUCACGAAA 20 downstream (SEQ ID NO: 2774)CEP290-1967 − UUCAAGCUAAUACAUGA 17 downstream (SEQ ID NO: 1004)CEP290-1968 + UUCCAUUCUUCACACAUGAA 20 downstream (SEQ ID NO: 2776)CEP290-1969 − UUGAGGUAGAAUCAAGA 17 downstream (SEQ ID NO: 2777)

Table 11 provides targeting domains for break-induced deletion ofgenomic sequence including the mutation at the LCA10 target position inthe CEP290 gene by dual targeting (e.g., dual double strand cleavage).Exemplary gRNA pairs to be used with S. aureus Cas9 are shown in Table11, e.g., CEP290-323 can be combined with CEP290-11, CEP290-323 can becombined with CEP290-64, CEP290-490 can be combined with CEP290-496,CEP290-490 can be combined with CEP290-502, CEP290-490 can be combinedwith CEP290-504, CEP290-492 can be combined with CEP290-502, orCEP290-492 can be combined with CEP290-504.

TABLE 11 Upstream gRNA (SEQ ID NO) Downstream gRNA (SEQ ID NO)CEP290-323 GTTCTGTCCTCAGTAAAAGGTA CEP290-11 GACACTGCCAATAGGG (SEQ ID NO:389) ATAGGT (corresponding RNA sequence in (SEQ ID NO: 387) SEQ ID NO:530) (corresponding RNA sequence in SEQ ID NO: 1047) CEP290-323GTTCTGTCCTCAGTAAAAGGTA CEP290-64 GTCAAAAGCTACCGGT (SEQ ID NO: 389)TACCTG (SEQ ID NO: 388) (corresponding RNA sequence in SEQ ID NO: 558)CEP290-490 GAATAGTTTGTTCTGGGTAC CEP290-496 GATGCAGAACTAGTGT (SEQ ID NO:390) AGAC (SEQ ID NO: 392) (corresponding RNA sequence in (correspondingRNA SEQ ID NO: 468) sequence in SEQ ID NO: 460) CEP290-490GAATAGTTTGTTCTGGGTAC CEP290-502 GTCACATGGGAGTCAC (SEQ ID NO: 390) AGGG(SEQ ID NO: 393) (corresponding RNA sequence in SEQ ID NO: 586)CEP290-490 GAATAGTTTGTTCTGGGTAC CEP290-504 GAGTATCTCCTGTTTGG (SEQ ID NO:390) CA (SEQ ID NO: 394) (corresponding RNA sequence in SEQ ID NO: 568)CEP290-492 GAGAAAGGGATGGGCACTTA CEP290-502 GTCACATGGGAGTCAC (SEQ ID NO:391) AGGG (corresponding RNA sequence in (SEQ ID NO: 393) SEQ ID NO:538) CEP290-492 GAGAAAGGGATGGGCACTTA CEP290-504 GAGTATCTCCTGTTTGG (SEQID NO: 391) CA (SEQ ID NO: 394)IV. RNA-Guided Nucleases

RNA-guided nucleases according to the present disclosure include,without limitation, naturally-occurring Class 2 CRISPR nucleases such asCas9, and Cpf1, as well as other nucleases derived or obtainedtherefrom. In functional terms, RNA-guided nucleases are defined asthose nucleases that: (a) interact with (e.g., complex with) a gRNA; and(b) together with the gRNA, associate with, and optionally cleave ormodify, a target region of a DNA that includes (i) a sequencecomplementary to the targeting domain of the gRNA and, optionally, (ii)an additional sequence referred to as a “protospacer adjacent motif,” or“PAM,” which is described in greater detail below. As the followingexamples will illustrate, RNA-guided nucleases can be defined, in broadterms, by their PAM specificity and cleavage activity, even thoughvariations may exist between individual RNA-guided nucleases that sharethe same PAM specificity or cleavage activity. Skilled artisans willappreciate that some aspects of the present disclosure relate tosystems, methods and compositions that can be implemented using anysuitable RNA-guided nuclease having a certain PAM specificity and/orcleavage activity. For this reason, unless otherwise specified, the termRNA-guided nuclease should be understood as a generic term, and notlimited to any particular type (e.g., Cas9 vs. Cpf1), species (e.g., S.pyogenes vs. S. aureus) or variation (e.g., full-length vs. truncated orsplit; naturally-occurring PAM specificity vs. engineered PAMspecificity).

Turning to the PAM sequence, this structure takes its name from itssequential relationship to the “protospacer” sequence that iscomplementary to gRNA targeting domains (or “spacers”). Together withprotospacer sequences, PAM sequences define target regions or sequencesfor specific RNA-guided nuclease/gRNA combinations.

Various RNA-guided nucleases may require different sequentialrelationships between PAMs and protospacers. In general, Cas9s recognizePAM sequences that are 5′ of the protospacer as visualized relative tothe top or complementary strand.

In addition to recognizing specific sequential orientations of PAMs andprotospacers, RNA-guided nucleases generally recognize specific PAMsequences. S. aureus Cas9, for example, recognizes a PAM sequence ofNNGRRT, wherein the N sequences are immediately 3′ of the regionrecognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGGPAM sequences. It should also be noted that engineered RNA-guidednucleases can have PAM specificities that differ from the PAMspecificities of similar nucleases (such as the naturally occurringvariant from which an RNA-guided nuclease is derived, or the naturallyoccurring variant having the greatest amino acid sequence homology to anengineered RNA-guided nuclease). Modified Cas9s that recognize alternatePAM sequences are described below.

RNA-guided nucleases are also characterized by their DNA cleavageactivity: naturally-occurring RNA-guided nucleases typically form DSBsin target nucleic acids, but engineered variants have been produced thatgenerate only SSBs (discussed above; see also Ran 2013, incorporated byreference herein), or that do not cut at all.

Cas9 Molecules

Crystal structures have been determined for S. pyogenes Cas9 (Jinek2014), and for S. aureus Cas9 in complex with a unimolecular gRNA and atarget DNA (Nishimasu 2014; Anders 2014; and Nishimasu 2015).

Cas9 molecules of a variety of species can be used in the methods andcompositions described herein. While the S. pyogenes, S. aureus, and S.thermophilus Cas9 molecules are the subject of much of the disclosureherein, Cas9 molecules of, derived from, or based on the Cas9 proteinsof other species listed herein can be used as well. In other words,while the much of the description herein uses S. pyogenes and S.thermophilus Cas9 molecules Cas9 molecules from the other species canreplace them. Such species include: Acidovorax avenae, Actinobacilluspleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis,Actinomyces sp., Cycliphilus denitrificans, Aminomonas paucivorans,Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroidessp., Blastopirellula marina, Bradyrhizobium sp., Brevibacilluslaterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacterlari, Candidatus puniceispirillum, Clostridium cellulolyticum,Clostridium perfringens, Corynebacterium accolens, Corynebacteriumdiphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae,Eubacterium dolichum, Gammaproteobacterium, Gluconacetobacterdiazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum,Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae,Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus,Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium,Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris,Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens,Neisseria lactamica, Neisseria meningitidis, Neisseria sp., Neisseriawadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurellamultocida, Phascolarctobacterium succinatutens, Ralstonia syzygii,Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri,Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus aureus,Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp.,Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae.

A Cas9 molecule, or Cas9 polypeptide, as that term is used herein,refers to a molecule or polypeptide that can interact with a guide RNA(gRNA) molecule and, in concert with the gRNA molecule, homes orlocalizes to a site which comprises a target domain and PAM sequence.Cas9 molecule and Cas9 polypeptide, as those terms are used herein,refer to naturally occurring Cas9 molecules and to engineered, altered,or modified Cas9 molecules or Cas9 polypeptides that differ, e.g., by atleast one amino acid residue, from a reference sequence, e.g., the mostsimilar naturally occurring Cas9 molecule or a sequence of Table 12.

Cas9 Domains

Crystal structures have been determined for two different naturallyoccurring bacterial Cas9 molecules (Jinek 2014) and for S. pyogenes Cas9with a guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA)(Nishimasu 2014; Anders 2014).

A naturally occurring Cas9 molecule comprises two lobes: a recognition(REC) lobe and a nuclease (NUC) lobe; each of which further comprisesdomains described herein. FIGS. 8A-8B provide a schematic of theorganization of important Cas9 domains in the primary structure. Thedomain nomenclature and the numbering of the amino acid residuesencompassed by each domain used throughout this disclosure is asdescribed in Nishimasu 2014. The numbering of the amino acid residues iswith reference to Cas9 from S. pyogenes.

The REC lobe comprises the arginine-rich bridge helix (BH), the REC1domain, and the REC2 domain. The REC lobe does not share structuralsimilarity with other known proteins, indicating that it is aCas9-specific functional domain. The BH domain is a long a helix andarginine rich region and comprises amino acids 60-93 of the sequence ofS. pyogenes Cas9. The REC1 domain is important for recognition of therepeat:anti-repeat duplex, e.g., of a gRNA or a tracrRNA, and istherefore critical for Cas9 activity by recognizing the target sequence.The REC1 domain comprises two REC1 motifs at amino acids 94 to 179 and308 to 717 of the sequence of S. pyogenes Cas9. These two REC1 domains,though separated by the REC2 domain in the linear primary structure,assemble in the tertiary structure to form the REC1 domain. The REC2domain, or parts thereof, may also play a role in the recognition of therepeat:anti-repeat duplex. The REC2 domain comprises amino acids 180-307of the sequence of S. pyogenes Cas9.

The NUC lobe comprises the RuvC domain (also referred to herein asRuvC-like domain), the HNH domain (also referred to herein as HNH-likedomain), and the PAM-interacting (PI) domain. The RuvC domain sharesstructural similarity to retroviral integrase superfamily members andcleaves a single strand, e.g., the non-complementary strand of thetarget nucleic acid molecule. The RuvC domain is assembled from thethree split RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are oftencommonly referred to in the art as RuvCI domain, or N-terminal RuvCdomain, RuvCII domain, and RuvCIII domain) at amino acids 1-59, 718-769,and 909-1098, respectively, of the sequence of S. pyogenes Cas9. Similarto the REC1 domain, the three RuvC motifs are linearly separated byother domains in the primary structure, however in the tertiarystructure, the three RuvC motifs assemble and form the RuvC domain. TheHNH domain shares structural similarity with HNH endonucleases, andcleaves a single strand, e.g., the complementary strand of the targetnucleic acid molecule. The HNH domain lies between the RuvC II-IIImotifs and comprises amino acids 775-908 of the sequence of S. pyogenesCas9. The PI domain interacts with the PAM of the target nucleic acidmolecule, and comprises amino acids 1099-1368 of the sequence of S.pyogenes Cas9.

RuvC-Like Domain and HNH-Like Domain

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises anHNH-like domain and a RuvC-like domain. In an embodiment, cleavageactivity is dependent on a RuvC-like domain and an HNH-like domain. ACas9 molecule or Cas9 polypeptide, e.g., an eaCas9 molecule or eaCas9polypeptide, can comprise one or more of the following domains: aRuvC-like domain and an HNH-like domain. In an embodiment, a Cas9molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptideand the eaCas9 molecule or eaCas9 polypeptide comprises a RuvC-likedomain, e.g., a RuvC-like domain described below, and/or an HNH-likedomain, e.g., an HNH-like domain described below.

RuvC-Like Domains

In an embodiment, a RuvC-like domain cleaves, a single strand, e.g., thenon-complementary strand of the target nucleic acid molecule. The Cas9molecule or Cas9 polypeptide can include more than one RuvC-like domain(e.g., one, two, three or more RuvC-like domains). In an embodiment, aRuvC-like domain is at least 5, 6, 7, 8 amino acids in length but notmore than 20, 19, 18, 17, 16 or 15 amino acids in length. In anembodiment, the Cas9 molecule or Cas9 polypeptide comprises anN-terminal RuvC-like domain of about 10 to 20 amino acids, e.g., about15 amino acids in length.

N-Terminal RuvC-Like Domains

Some naturally occurring Cas9 molecules comprise more than one RuvC-likedomain with cleavage being dependent on the N-terminal RuvC-like domain.Accordingly, Cas9 molecules or Cas9 polypeptide can comprise anN-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains aredescribed below.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises anN-terminal RuvC-like domain comprising an amino acid sequence of formulaI:

(SEQ ID NO: 8) D-X1-G-X2-X3-X4-X5-G-X6-X7-X8-X9,

wherein,

X1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);

X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V,and I);

X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);

X4 is selected from S, Y, N and F (e.g., S);

X5 is selected from V, I, L, C, T and F (e.g., selected from V, I andL);

X6 is selected from W, F, V, Y, S and L (e.g., W);

X7 is selected from A, S, C, V and G (e.g., selected from A and S);

X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M andL); and

X9 is selected from any amino acid or is absent, designated by Δ (e.g.,selected from T, V, I, L, Δ, F, S, A, Y, M and R, or, e.g., selectedfrom T, V, I, L and Δ).

In an embodiment, the N-terminal RuvC-like domain differs from asequence of SEQ ID NO: 8, by as many as 1 but no more than 2, 3, 4, or 5residues.

In embodiment, the N-terminal RuvC-like domain is cleavage competent.

In embodiment, the N-terminal RuvC-like domain is cleavage incompetent.

In an embodiment, a eaCas9 molecule or eaCas9 polypeptide comprises anN-terminal RuvC-like domain comprising an amino acid sequence of formulaII:

(SEQ ID NO: 9) D-X1-G-X2-X3-S-X5-G-X6-X7-X8-X9,,

wherein

X1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);

X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V,and I);

X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);

X5 is selected from V, I, L, C, T and F (e.g., selected from V, I andL);

X6 is selected from W, F, V, Y, S and L (e.g., W);

X7 is selected from A, S, C, V and G (e.g., selected from A and S);

X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M andL); and

X9 is selected from any amino acid or is absent (e.g., selected from T,V, I, L, Δ, F, S, A, Y, M and R or selected from e.g., T, V, I, L andΔ).

In an embodiment, the N-terminal RuvC-like domain differs from asequence of SEQ ID NO:9 by as many as 1 but no more than 2, 3, 4, or 5residues.

In an embodiment, the N-terminal RuvC-like domain comprises an aminoacid sequence of formula III:

(SEQ ID NO: 10) D-I-G-X2-X3-S-V-G-W-A-X8-X9,

wherein

X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V,and I);

X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);

X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M andL); and

X9 is selected from any amino acid or is absent (e.g., selected from T,V, I, L, Δ, F, S, A, Y, M and R or selected from e.g., T, V, I, L andΔ).

In an embodiment, the N-terminal RuvC-like domain differs from asequence of SEQ ID NO: 10 by as many as 1 but no more than, 2, 3, 4, or5 residues.

In an embodiment, the N-terminal RuvC-like domain comprises an aminoacid sequence of formula III:

(SEQ ID NO: 11) D-I-G-T-N-S-V-G-W-A-V-X,

wherein

X is a non-polar alkyl amino acid or a hydroxyl amino acid, e.g., X isselected from V, I, L and T (e.g., the eaCas9 molecule can comprise anN-terminal RuvC-like domain shown in FIGS. 2A-2G (is depicted as Y)).

In an embodiment, the N-terminal RuvC-like domain differs from asequence of SEQ ID NO: 11 by as many as 1 but no more than, 2, 3, 4, or5 residues.

In an embodiment, the N-terminal RuvC-like domain differs from asequence of an N-terminal RuvC like domain disclosed herein, e.g., inFIGS. 3A-3B or FIGS. 7A-7B, as many as 1 but no more than 2, 3, 4, or 5residues. In an embodiment, 1, 2, or all 3 of the highly conservedresidues identified in FIGS. 3A-3B or FIGS. 7A-7B are present.

In an embodiment, the N-terminal RuvC-like domain differs from asequence of an N-terminal RuvC-like domain disclosed herein, e.g., inFIGS. 4A-4B or FIGS. 7A-7B, as many as 1 but no more than 2, 3, 4, or 5residues. In an embodiment, 1, 2, 3 or all 4 of the highly conservedresidues identified in FIGS. 4A-4B or FIGS. 7A-7B are present.

Additional RuvC-Like Domains

In addition to the N-terminal RuvC-like domain, the Cas9 molecule orCas9 polypeptide, e.g., an eaCas9 molecule or eaCas9 polypeptide, cancomprise one or more additional RuvC-like domains. In an embodiment, theCas9 molecule or Cas9 polypeptide can comprise two additional RuvC-likedomains. Preferably, the additional RuvC-like domain is at least 5 aminoacids in length and, e.g., less than 15 amino acids in length, e.g., 5to 10 amino acids in length, e.g., 8 amino acids in length.

An additional RuvC-like domain can comprise an amino acid sequence:

(SEQ ID NO: 12) I-X1-X2-E-X3-A-R-E,wherein

X1 is V or H,

X2 is I, L or V (e.g., I or V); and

X3 is M or T.

In an embodiment, the additional RuvC-like domain comprises the aminoacid sequence:I-V-X2-E-M-A-R-E  (SEQ ID NO: 13),wherein

X2 is I, L or V (e.g., I or V) (e.g., the eaCas9 molecule or eaCas9polypeptide can comprise an additional RuvC-like domain shown in FIG.2A-2G or FIGS. 7A-7B (depicted as B)).

An additional RuvC-like domain can comprise an amino acid sequence:

(SEQ ID NO: 14) H-H-A-X1-D-A-X2-X3,

X1 is H or L;

X2 is R or V; and

X3 is E or V.

In an embodiment, the additional RuvC-like domain comprises the aminoacid sequence:H-H-A-H-D-A-Y-L  (SEQ ID NO: 15).

In an embodiment, the additional RuvC-like domain differs from asequence of SEQ ID NOs: 13, 15, 12 or 14 by as many as 1 but no morethan 2, 3, 4, or 5 residues.

In some embodiments, the sequence flanking the N-terminal RuvC-likedomain is a sequences of formula V:

(SEQ ID NO: 16) K-X1′-Y-X2′-X3′-X4′-Z-T-D-X9′-Y,.

wherein

X1′ is selected from K and P,

X2′ is selected from V, L, I, and F (e.g., V, I and L);

X3′ is selected from G, A and S (e.g., G),

X4′ is selected from L, I, V and F (e.g., L);

X9′ is selected from D, E, N and Q; and

Z is an N-terminal RuvC-like domain, e.g., as described above.

HNH-Like Domains

In an embodiment, an HNH-like domain cleaves a single strandedcomplementary domain, e.g., a complementary strand of a double strandednucleic acid molecule. In an embodiment, an HNH-like domain is at least15, 20, 25 amino acids in length but not more than 40, 35 or 30 aminoacids in length, e.g., 20 to 35 amino acids in length, e.g., 25 to 30amino acids in length. Exemplary HNH-like domains are described below.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises anHNH-like domain having an amino acid sequence of formula VI:

(SEQ ID NO: 17) X1-X2-X3-H-X4-X5-P-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-N-X16-X17-X18-X19-X20-X21-X22-X23-N,wherein

X1 is selected from D, E, Q and N (e.g., D and E);

X2 is selected from L, I, R, Q, V, M and K;

X3 is selected from D and E;

X4 is selected from I, V, T, A and L (e.g., A, I and V);

X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);

X6 is selected from Q, H, R, K, Y, I, L, F and W;

X7 is selected from S, A, D, T and K (e.g., S and A);

X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;

X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

X11 is selected from D, S, N, R, L and T (e.g., D);

X12 is selected from D, N and S;

X13 is selected from S, A, T, G and R (e.g., S);

X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L andF);

X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X16 is selected from K, L, R, M, T and F (e.g., L, R and K);

X17 is selected from V, L, I, A and T;

X18 is selected from L, I, V and A (e.g., L and I);

X19 is selected from T, V, C, E, S and A (e.g., T and V);

X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;

X21 is selected from S, P, R, K, N, A, H, Q, G and L;

X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and

X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In an embodiment, a HNH-like domain differs from a sequence of SEQ IDNO: 16 by at least one but no more than, 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain is cleavage competent.

In an embodiment, the HNH-like domain is cleavage incompetent.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises anHNH-like domain comprising an amino acid sequence of formula VII:

(SEQ ID NO: 18) X1-X2-X3-H-X4-X5-P-X6-S-X8-X9-X10-D-D-S-X14-X15-N-K-V-L-X19-X20-X21-X22-X23-N,

wherein

X1 is selected from D and E;

X2 is selected from L, I, R, Q, V, M and K;

X3 is selected from D and E;

X4 is selected from I, V, T, A and L (e.g., A, I and V);

X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);

X6 is selected from Q, H, R, K, Y, I, L, F and W;

X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;

X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L andF);

X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X19 is selected from T, V, C, E, S and A (e.g., T and V);

X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;

X21 is selected from S, P, R, K, N, A, H, Q, G and L;

X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and

X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In an embodiment, the HNH-like domain differs from a sequence of SEQ IDNO: 15 by 1, 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises anHNH-like domain comprising an amino acid sequence of formula VII:

(SEQ ID NO: 19) X1-V-X3-H-I-V-P-X6-S-X8-X9-X10-D-D-S-X14-X15-N-K-V-L-T-X20-X21-X22-X23-N,

wherein

X1 is selected from D and E;

X3 is selected from D and E;

X6 is selected from Q, H, R, K, Y, I, L and W;

X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);

X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;

X10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;

X14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L andF);

X15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;

X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;

X21 is selected from S, P, R, K, N, A, H, Q, G and L;

X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and

X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.

In an embodiment, the HNH-like domain differs from a sequence of SEQ IDNO:GG by 1, 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises anHNH-like domain having an amino acid sequence of formula VIII:

(SEQ ID NO: 20) D-X2-D-H-I-X5-P-Q-X7-F-X9-X10-D-X12-S-I-D-N-X16-V-L-X19-X20-S-X22-X23-N,

wherein

X2 is selected from I and V;

X5 is selected from I and V;

X7 is selected from A and S;

X9 is selected from I and L;

X10 is selected from K and T;

X12 is selected from D and N;

X16 is selected from R, K and L; X19 is selected from T and V;

X20 is selected from S and R;

X22 is selected from K, D and A; and

X23 is selected from E, K, G and N (e.g., the eaCas9 molecule or eaCas9polypeptide can comprise an HNH-like domain as described herein).

In an embodiment, the HNH-like domain differs from a sequence of SEQ IDNO: 19 by as many as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises theamino acid sequence of formula IX:

(SEQ ID NO: 21) L-Y-Y-L-Q-N-G-X1′-D-M-Y-X2′-X3′-X4′-X5′-L-D-I-X6′-X7′-L-S-X8′-Y-Z-N-R-X9′-K-X10′-D-X11′-V-P,

wherein

X1′ is selected from K and R;

X2′ is selected from V and T;

X3′ is selected from G and D;

X4′ is selected from E, Q and D;

X5′ is selected from E and D;

X6′ is selected from D, N and H;

X7′ is selected from Y, R and N;

X8′ is selected from Q, D and N; X9′ is selected from G and E;

X10′ is selected from S and G;

X11′ is selected from D and N; and

Z is an HNH-like domain, e.g., as described above.

In an embodiment, the eaCas9 molecule or eaCas9 polypeptide comprises anamino acid sequence that differs from a sequence of SEQ ID NO: 21 by asmany as 1 but no more than 2, 3, 4, or 5 residues.

In an embodiment, the HNH-like domain differs from a sequence of anHNH-like domain disclosed herein, e.g., in FIGS. 5A-5C or FIGS. 7A-7B,as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment,1 or both of the highly conserved residues identified in FIGS. 5A-5C orFIGS. 7A-7B are present.

In an embodiment, the HNH-like domain differs from a sequence of anHNH-like domain disclosed herein, e.g., in FIGS. 6A-6B or FIGS. 7A-7B,as many as 1 but no more than 2, 3, 4, or 5 residues. In an embodiment,1, 2, all 3 of the highly conserved residues identified in FIGS. 6A-6Bor FIGS. 7A-7B are present.

Cas9 Activities

Nuclease and Helicase Activities

In an embodiment, the Cas9 molecule or Cas9 polypeptide is capable ofcleaving a target nucleic acid molecule. Typically wild type Cas9molecules cleave both strands of a target nucleic acid molecule. Cas9molecules and Cas9 polypeptides can be engineered to alter nucleasecleavage (or other properties), e.g., to provide a Cas9 molecule or Cas9polypeptide which is a nickase, or which lacks the ability to cleavetarget nucleic acid. A Cas9 molecule or Cas9 polypeptide that is capableof cleaving a target nucleic acid molecule is referred to herein as aneaCas9 molecule or eaCas9 polypeptide.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises oneor more of the following activities:

a nickase activity, i.e., the ability to cleave a single strand, e.g.,the non-complementary strand or the complementary strand, of a nucleicacid molecule;

a double stranded nuclease activity, i.e., the ability to cleave bothstrands of a double stranded nucleic acid and create a double strandedbreak, which in an embodiment is the presence of two nickase activities;

an endonuclease activity;

an exonuclease activity; and

a helicase activity, i.e., the ability to unwind the helical structureof a double stranded nucleic acid.

In an embodiment, an enzymatically active or eaCas9 molecule or eaCas9polypeptide cleaves both strands and results in a double stranded break.In an embodiment, an eaCas9 molecule cleaves only one strand, e.g., thestrand to which the gRNA hybridizes to, or the strand complementary tothe strand the gRNA hybridizes with. In an embodiment, an eaCas9molecule or eaCas9 polypeptide comprises cleavage activity associatedwith an HNH-like domain. In an embodiment, an eaCas9 molecule or eaCas9polypeptide comprises cleavage activity associated with an N-terminalRuvC-like domain. In an embodiment, an eaCas9 molecule or eaCas9polypeptide comprises cleavage activity associated with an HNH-likedomain and cleavage activity associated with an N-terminal RuvC-likedomain. In an embodiment, an eaCas9 molecule or eaCas9 polypeptidecomprises an active, or cleavage competent, HNH-like domain and aninactive, or cleavage incompetent, N-terminal RuvC-like domain. In anembodiment, an eaCas9 molecule or eaCas9 polypeptide comprises aninactive, or cleavage incompetent, HNH-like domain and an active, orcleavage competent, N-terminal RuvC-like domain.

Some Cas9 molecules or Cas9 polypeptides have the ability to interactwith a gRNA molecule, and in conjunction with the gRNA molecule localizeto a core target domain, but are incapable of cleaving the targetnucleic acid, or incapable of cleaving at efficient rates. Cas9molecules having no, or no substantial, cleavage activity are referredto herein as an eiCas9 molecule or eiCas9 polypeptide. For example, aneiCas9 molecule or eiCas9 polypeptide can lack cleavage activity or havesubstantially less, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavageactivity of a reference Cas9 molecule or eiCas9 polypeptide, as measuredby an assay described herein.

Targeting and PAMs

RNA guided nucleases, such as Cas9 molecules or Cas9 polypeptides,generally, interact with a guide RNA (gRNA) molecule and, in concertwith the gRNA molecule, localize to a site which comprises a targetdomain and a PAM sequence.

In an embodiment, the ability of an eaCas9 molecule or eaCas9polypeptide to interact with and cleave a target nucleic acid is PAMsequence dependent. A PAM sequence is a sequence in the target nucleicacid. In an embodiment, cleavage of the target nucleic acid occursupstream from the PAM sequence. EaCas9 molecules from differentbacterial species can recognize different sequence motifs (e.g., PAMsequences). In an embodiment, an eaCas9 molecule of S. pyogenesrecognizes the sequence motif NGG, NAG, NGA and directs cleavage of atarget nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstreamfrom that sequence. See, e.g., Mali 2013. In an embodiment, an eaCas9molecule of S. thermophilus recognizes the sequence motif NGGNG andNNAGAAW (W=A or T) and directs cleavage of a core target nucleic acidsequence 1 to 10, e.g., 3 to 5, base pairs upstream from thesesequences. See, e.g., Horvath 2010 and Deveau 2008. In an embodiment, aneaCas9 molecule of S. mutans recognizes the sequence motif NGG and/orNAAR (R=A or G) and directs cleavage of a core target nucleic acidsequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence.See, e.g., Deveau 2008. In an embodiment, an eaCas9 molecule of S.aureus recognizes the sequence motif NNGRR (R=A or G) and directscleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, basepairs upstream from that sequence. In an embodiment, an eaCas9 moleculeof S. aureus recognizes the sequence motif NNGRRN (R=A or G) and directscleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, basepairs upstream from that sequence. In an embodiment, an eaCas9 moleculeof S. aureus recognizes the sequence motif NNGRRT (R=A or G) and directscleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, basepairs upstream from that sequence. In an embodiment, an eaCas9 moleculeof S. aureus recognizes the sequence motif NNGRRV (R=A or G, V=A, G orC) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g.,3 to 5, base pairs upstream from that sequence. In an embodiment, aneaCas9 molecule of Neisseria meningitidis recognizes the sequence motifNNNNGATT or NNNGCTT and directs cleavage of a target nucleic acidsequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence.See, e.g., Hou 2013. The ability of a Cas9 molecule to recognize a PAMsequence can be determined, e.g., using a transformation assay describedin Jinek 2012. In the aforementioned embodiments, N can be anynucleotide residue, e.g., any of A, G, C or T.

As is discussed herein, Cas9 molecules can be engineered to alter thePAM specificity of the Cas9 molecule.

Exemplary naturally occurring Cas9 molecules are described in Chylinski2013. Such Cas9 molecules include Cas9 molecules of a cluster 1bacterial family, cluster 2 bacterial family, cluster 3 bacterialfamily, cluster 4 bacterial family, cluster 5 bacterial family, cluster6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterialfamily, a cluster 9 bacterial family, a cluster 10 bacterial family, acluster 11 bacterial family, a cluster 12 bacterial family, a cluster 13bacterial family, a cluster 14 bacterial family, a cluster 15 bacterialfamily, a cluster 16 bacterial family, a cluster 17 bacterial family, acluster 18 bacterial family, a cluster 19 bacterial family, a cluster 20bacterial family, a cluster 21 bacterial family, a cluster 22 bacterialfamily, a cluster 23 bacterial family, a cluster 24 bacterial family, acluster 25 bacterial family, a cluster 26 bacterial family, a cluster 27bacterial family, a cluster 28 bacterial family, a cluster 29 bacterialfamily, a cluster 30 bacterial family, a cluster 31 bacterial family, acluster 32 bacterial family, a cluster 33 bacterial family, a cluster 34bacterial family, a cluster 35 bacterial family, a cluster 36 bacterialfamily, a cluster 37 bacterial family, a cluster 38 bacterial family, acluster 39 bacterial family, a cluster 40 bacterial family, a cluster 41bacterial family, a cluster 42 bacterial family, a cluster 43 bacterialfamily, a cluster 44 bacterial family, a cluster 45 bacterial family, acluster 46 bacterial family, a cluster 47 bacterial family, a cluster 48bacterial family, a cluster 49 bacterial family, a cluster 50 bacterialfamily, a cluster 51 bacterial family, a cluster 52 bacterial family, acluster 53 bacterial family, a cluster 54 bacterial family, a cluster 55bacterial family, a cluster 56 bacterial family, a cluster 57 bacterialfamily, a cluster 58 bacterial family, a cluster 59 bacterial family, acluster 60 bacterial family, a cluster 61 bacterial family, a cluster 62bacterial family, a cluster 63 bacterial family, a cluster 64 bacterialfamily, a cluster 65 bacterial family, a cluster 66 bacterial family, acluster 67 bacterial family, a cluster 68 bacterial family, a cluster 69bacterial family, a cluster 70 bacterial family, a cluster 71 bacterialfamily, a cluster 72 bacterial family, a cluster 73 bacterial family, acluster 74 bacterial family, a cluster 75 bacterial family, a cluster 76bacterial family, a cluster 77 bacterial family, or a cluster 78bacterial family.

Exemplary naturally occurring Cas9 molecules include a Cas9 molecule ofa cluster 1 bacterial family. Examples include a Cas9 molecule of: S.pyogenes (e.g., strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315,MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (e.g.,strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. mutans(e.g., strain UA159, NN2025), S. macacae (e.g., strain NCTC11558), S.gallolyticus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g.,strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S.bovis (e.g., strain ATCC 700338), S. anginosus (e.g., strain F0211), S.agalactiae (e.g., strain NEM316, A909), Listeria monocytogenes (e.g.,strain F6854), Listeria innocua (L. innocua, e.g., strain Clip 11262),Enterococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium(e.g., strain 1,231,408). Another exemplary Cas9 molecule is a Cas9molecule of Neisseria meningitidis (Hou 2013).

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9molecule or eaCas9 polypeptide, comprises an amino acid sequence:

having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%homology with;

differs at no more than, 2, 5, 10, 15, 20, 30, or 40% of the amino acidresidues when compared with;

differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than100, 80, 70, 60, 50, 40 or 30 amino acids from; or

is identical to any Cas9 molecule sequence described herein, or anaturally occurring Cas9 molecule sequence, e.g., a Cas9 molecule from aspecies listed herein or described in Chylinski 2013; Hou 2013; SEQ IDNOs: 1-4. In an embodiment, the Cas9 molecule or Cas9 polypeptidecomprises one or more of the following activities: a nickase activity; adouble stranded cleavage activity (e.g., an endonuclease and/orexonuclease activity); a helicase activity; or the ability, togetherwith a gRNA molecule, to home to a target nucleic acid.

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises theamino acid sequence of the consensus sequence of FIGS. 2A-2G, wherein“*” indicates any amino acid found in the corresponding position in theamino acid sequence of a Cas9 molecule of S. pyogenes, S. thermophilus,S. mutans and L. innocua, and “-” indicates any amino acid. In anembodiment, a Cas9 molecule or Cas9 polypeptide differs from thesequence of the consensus sequence disclosed in FIGS. 2A-2G by at least1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises theamino acid sequence of SEQ ID NO: 7 of FIGS. 7A-7B, wherein “*”indicates any amino acid found in the corresponding position in theamino acid sequence of a Cas9 molecule of S. pyogenes, or N.meningitidis, “-” indicates any amino acid, and “-” indicates any aminoacid or absent. In an embodiment, a Cas9 molecule or Cas9 polypeptidediffers from the sequence of SEQ ID NOs: 6 or 7 disclosed in FIGS. 7A-7Bby at least 1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidresidues.

A comparison of the sequence of a number of Cas9 molecules indicate thatcertain regions are conserved. These are identified below as:

region 1 (residues 1 to 180, or in the case of region 1′ residues 120 to180) region 2 (residues 360 to 480);

region 3 (residues 660 to 720);

region 4 (residues 817 to 900); and

region 5 (residues 900 to 960);

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises regions1-5, together with sufficient additional Cas9 molecule sequence toprovide a biologically active molecule, e.g., a Cas9 molecule having atleast one activity described herein. In an embodiment, each of regions1-6, independently, have, 50%, 60%, 70%, or 80% homology with thecorresponding residues of a Cas9 molecule or Cas9 polypeptide describedherein, e.g., a sequence from FIGS. 2A-2G or from FIGS. 7A-7B.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9molecule or eaCas9 polypeptide, comprises an amino acid sequencereferred to as region 1:

having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologywith amino acids 1-180 (the numbering is according to the motif sequencein FIGS. 2A-2G; 52% of residues in the four Cas9 sequences in FIGS.2A-2G are conserved) of the amino acid sequence of Cas9 of S. pyogenes;

differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than90, 80, 70, 60, 50, 40 or 30 amino acids from amino acids 1-180 of theamino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutansor L. innocua; or

is identical to 1-180 of the amino acid sequence of Cas9 of S. pyogenes,S. thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9molecule or eaCas9 polypeptide, comprises an amino acid sequencereferred to as region 1′:

having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%homology with amino acids 120-180 (55% of residues in the four Cas9sequences in FIGS. 2A-2G are conserved) of the amino acid sequence ofCas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 120-180 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L.innocua; or

is identical to 120-180 of the amino acid sequence of Cas9 of S.pyogenes, S. thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9molecule or eaCas9 polypeptide, comprises an amino acid sequencereferred to as region 2:

having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%or 99% homology with amino acids 360-480 (52% of residues in the fourCas9 sequences in FIGS. 2A-2G are conserved) of the amino acid sequenceof Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 360-480 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L.innocua; or

is identical to 360-480 of the amino acid sequence of Cas9 of S.pyogenes, S. thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9molecule or eaCas9 polypeptide, comprises an amino acid sequencereferred to as region 3:

having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% homology with amino acids 660-720 (56% of residues in the four Cas9sequences in FIGS. 2A-2G are conserved) of the amino acid sequence ofCas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 660-720 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L.innocua; or

is identical to 660-720 of the amino acid sequence of Cas9 of S.pyogenes, S. thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9molecule or eaCas9 polypeptide, comprises an amino acid sequencereferred to as region 4:

having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% homology with amino acids 817-900 (55% of residues in the fourCas9 sequences in FIGS. 2A-2G are conserved) of the amino acid sequenceof Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 817-900 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L.innocua; or

is identical to 817-900 of the amino acid sequence of Cas9 of S.pyogenes, S. thermophilus, S. mutans or L. innocua.

In an embodiment, a Cas9 molecule or Cas9 polypeptide, e.g., an eaCas9molecule or eaCas9 polypeptide, comprises an amino acid sequencereferred to as region 5:

having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% homology with amino acids 900-960 (60% of residues in the fourCas9 sequences in FIGS. 2A-2G are conserved) of the amino acid sequenceof Cas9 of S. pyogenes, S. thermophilus, S. mutans or L. innocua;

differs by at least 1, 2, or 5 amino acids but by no more than 35, 30,25, 20 or 10 amino acids from amino acids 900-960 of the amino acidsequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or L.innocua; or

is identical to 900-960 of the amino acid sequence of Cas9 of S.pyogenes, S. thermophilus, S. mutans or L. innocua.

Modifications of RNA-Guided Nucleases

The RNA-guided nucleases described above have activities and propertiesthat are useful in a variety of applications, but the skilled artisanwill appreciate that RNA-guided nucleases may also be modified incertain instances, to alter cleavage activity, PAM specificity, or otherstructural or functional features.

Turning first to modifications that alter cleavage activity, mutationsthat reduce or eliminate the activity of domains within the NUC lobehave been described above. As discussed in more detail below, exemplarymutations that may be made in the RuvC domains, in the Cas9 HNH domain,or in the Cpf1 Nuc domain are described in Ran and Yamano, as well as inCotta-Ramusino. In general, mutations that reduce or eliminate activityin one of the two nuclease domains result in RNA-guided nucleases withnickase activity, but it should be noted that the type of nickaseactivity varies depending on which domain is inactivated. As oneexample, inactivation of a RuvC domain of a Cas9 will result in anickase that cleaves the complementary strand, while inactivation of aCas9 HNH domain results in a nickase that cleaves the non-complementarystrand.

Modifications of PAM specificity relative to naturally occurring Cas9reference molecules has been described for both S. pyogenes (Kleinstiver2015a) and S. aureus (Kleinstiver 2015b). Modifications that improve thetargeting fidelity of Cas9 have also been described (Kleinstiver 2016).Each of these references is incorporated by reference herein.

RNA-guided nucleases have been split into two or more parts (see, e.g.,Zetsche 2015; Fine 2015; both incorporated by reference).

RNA-guided nucleases are, in some cases, size-optimized or truncated,for example via one or more deletions that reduce the size of thenuclease while still retaining gRNA association, target and PAMrecognition, and cleavage activities. In certain embodiments, RNA guidednucleases are bound, covalently or non-covalently, to anotherpolypeptide, nucleotide, or other structure, optionally by means of alinker. RNA-guided nucleases also optionally include a tag, such as anuclear localization signal to facilitate movement of RNA-guidednuclease protein into the nucleus.

Engineered or Altered Cas9 Molecules and Cas9 Polypeptides

Cas9 molecules and Cas9 polypeptides described herein, e.g., naturallyoccurring Cas9 molecules, can possess any of a number of properties,including: nickase activity, nuclease activity (e.g., endonucleaseand/or exonuclease activity); helicase activity; the ability toassociate functionally with a gRNA molecule; and the ability to target(or localize to) a site on a nucleic acid (e.g., PAM recognition andspecificity). In an embodiment, a Cas9 molecule or Cas9 polypeptide caninclude all or a subset of these properties. In typical embodiments, aCas9 molecule or Cas9 polypeptide has the ability to interact with agRNA molecule and, in concert with the gRNA molecule, localize to a sitein a nucleic acid. Other activities, e.g., PAM specificity, cleavageactivity, or helicase activity can vary more widely in Cas9 moleculesand Cas9 polypeptides.

Cas9 molecules include engineered Cas9 molecules and engineered Cas9polypeptides (engineered, as used in this context, means merely that theCas9 molecule or Cas9 polypeptide differs from a reference sequences,and implies no process or origin limitation). An engineered Cas9molecule or Cas9 polypeptide can comprise altered enzymatic properties,e.g., altered nuclease activity, (as compared with a naturally occurringor other reference Cas9 molecule) or altered helicase activity. Asdiscussed herein, an engineered Cas9 molecule or Cas9 polypeptide canhave nickase activity (as opposed to double strand nuclease activity).In an embodiment an engineered Cas9 molecule or Cas9 polypeptide canhave an alteration that alters its size, e.g., a deletion of amino acidsequence that reduces its size, e.g., without significant effect on oneor more, or any Cas9 activity. In an embodiment, an engineered Cas9molecule or Cas9 polypeptide can comprise an alteration that affects PAMrecognition. E.g., an engineered Cas9 molecule can be altered torecognize a PAM sequence other than that recognized by the endogenouswild-type PI domain. In an embodiment, a Cas9 molecule or Cas9polypeptide can differ in sequence from a naturally occurring Cas9molecule but not have significant alteration in one or more Cas9activities.

Cas9 molecules or Cas9 polypeptides with desired properties can be madein a number of ways, e.g., by alteration of a parental, e.g., naturallyoccurring, Cas9 molecules or Cas9 polypeptides, to provide an alteredCas9 molecule or Cas9 polypeptide having a desired property. Forexample, one or more mutations or differences relative to a parentalCas9 molecule, e.g., a naturally occurring or engineered Cas9 molecule,can be introduced. Such mutations and differences comprise:substitutions (e.g., conservative substitutions or substitutions ofnon-essential amino acids); insertions; or deletions. In an embodiment,a Cas9 molecule or Cas9 polypeptide can comprises one or more mutationsor differences, e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50mutations, but less than 200, 100, or 80 mutations relative to areference, e.g., a parental, Cas9 molecule.

In an embodiment, a mutation or mutations do not have a substantialeffect on a Cas9 activity, e.g. a Cas9 activity described herein. In anembodiment, a mutation or mutations have a substantial effect on a Cas9activity, e.g. a Cas9 activity described herein.

Non-Cleaving and Modified-Cleavage Cas9 Molecules and Cas9 Polypeptides

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises acleavage property that differs from naturally occurring Cas9 molecules,e.g., that differs from the naturally occurring Cas9 molecule having theclosest homology. For example, a Cas9 molecule or Cas9 polypeptide candiffer from naturally occurring Cas9 molecules, e.g., a Cas9 molecule ofS. pyogenes, as follows: its ability to modulate, e.g., decreased orincreased, cleavage of a double stranded nucleic acid (endonucleaseand/or exonuclease activity), e.g., as compared to a naturally occurringCas9 molecule (e.g., a Cas9 molecule of S. pyogenes); its ability tomodulate, e.g., decreased or increased, cleavage of a single strand of anucleic acid, e.g., a non-complementary strand of a nucleic acidmolecule or a complementary strand of a nucleic acid molecule (nickaseactivity), e.g., as compared to a naturally occurring Cas9 molecule(e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave anucleic acid molecule, e.g., a double stranded or single strandednucleic acid molecule, can be eliminated.

Modified Cleavage eaCas9 Molecules and eaCas9 Polypeptides

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises oneor more of the following activities: cleavage activity associated withan N-terminal RuvC-like domain; cleavage activity associated with anHNH-like domain; cleavage activity associated with an HNH-like domainand cleavage activity associated with an N-terminal RuvC-like domain.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises anactive, or cleavage competent, HNH-like domain (e.g., an HNH-like domaindescribed herein, e.g., SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQID NO: 20, or SEQ ID NO: 21) and an inactive, or cleavage incompetent,N-terminal RuvC-like domain. An exemplary inactive, or cleavageincompetent N-terminal RuvC-like domain can have a mutation of anaspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acidat position 9 of the consensus sequence disclosed in FIGS. 2A-2G or anaspartic acid at position 10 of SEQ ID NO: 7, e.g., can be substitutedwith an alanine. In an embodiment, the eaCas9 molecule or eaCas9polypeptide differs from wild type in the N-terminal RuvC-like domainand does not cleave the target nucleic acid, or cleaves withsignificantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% ofthe cleavage activity of a reference Cas9 molecule, e.g., as measured byan assay described herein. The reference Cas9 molecule can by anaturally occurring unmodified Cas9 molecule, e.g., a naturallyoccurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S.thermophilus. In an embodiment, the reference Cas9 molecule is thenaturally occurring Cas9 molecule having the closest sequence identityor homology.

In an embodiment, an eaCas9 molecule or eaCas9 polypeptide comprises aninactive, or cleavage incompetent, HNH domain and an active, or cleavagecompetent, N-terminal RuvC-like domain (e.g., an N-terminal RuvC-likedomain described herein, e.g., SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, or SEQ ID NO: 16). Exemplary inactive, or cleavage incompetentHNH-like domains can have a mutation at one or more of: a histidine inan HNH-like domain, e.g., a histidine shown at position 856 of FIGS.2A-2G, e.g., can be substituted with an alanine; and one or moreasparagines in an HNH-like domain, e.g., an asparagine shown at position870 of FIGS. 2A-2G and/or at position 879 of FIGS. 2A-2G, e.g., can besubstituted with an alanine. In an embodiment, the eaCas9 differs fromwild type in the HNH-like domain and does not cleave the target nucleicacid, or cleaves with significantly less efficiency, e.g., less than 20,10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule,e.g., as measured by an assay described herein. The reference Cas9molecule can by a naturally occurring unmodified Cas9 molecule, e.g., anaturally occurring Cas9 molecule such as a Cas9 molecule of S.pyogenes, or S. thermophilus. In an embodiment, the reference Cas9molecule is the naturally occurring Cas9 molecule having the closestsequence identity or homology.

Alterations in the Ability to Cleave One or Both Strands of a TargetNucleic Acid

In an embodiment, exemplary Cas9 activities comprise one or more of PAMspecificity, cleavage activity, and helicase activity. A mutation(s) canbe present, e.g., in one or more RuvC-like domain, e.g., an N-terminalRuvC-like domain; an HNH-like domain; a region outside the RuvC-likedomains and the HNH-like domain. In some embodiments, a mutation(s) ispresent in a RuvC-like domain, e.g., an N-terminal RuvC-like domain. Insome embodiments, a mutation(s) is present in an HNH-like domain. Insome embodiments, mutations are present in both a RuvC-like domain,e.g., an N-terminal RuvC-like domain, and an HNH-like domain.

Exemplary mutations that may be made in the RuvC domain or HNH domainwith reference to the S. pyogenes sequence include: D10A, E762A, H840A,N854A, N863A and/or D986A.

In an embodiment, a Cas9 molecule or Cas9 polypeptide is an eiCas9molecule or eiCas9 polypeptide comprising one or more differences in aRuvC domain and/or in an HNH domain as compared to a reference Cas9molecule, and the eiCas9 molecule or eiCas9 polypeptide does not cleavea nucleic acid, or cleaves with significantly less efficiency than doeswildtype, e.g., when compared with wild type in a cleavage assay, e.g.,as described herein, cuts with less than 50, 25, 10, or 1% of areference Cas9 molecule, as measured by an assay described herein.

Whether or not a particular sequence, e.g., a substitution, may affectone or more activity, such as targeting activity, cleavage activity,etc., can be evaluated or predicted, e.g., by evaluating whether themutation is conservative or by the method described in Section V. In anembodiment, a “non-essential” amino acid residue, as used in the contextof a Cas9 molecule, is a residue that can be altered from the wild-typesequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule,e.g., an eaCas9 molecule, without abolishing or more preferably, withoutsubstantially altering a Cas9 activity (e.g., cleavage activity),whereas changing an “essential” amino acid residue results in asubstantial loss of activity (e.g., cleavage activity).

In an embodiment, a Cas9 molecule or Cas9 polypeptide comprises acleavage property that differs from naturally occurring Cas9 molecules,e.g., that differs from the naturally occurring Cas9 molecule having theclosest homology. For example, a Cas9 molecule or Cas9 polypeptide candiffer from naturally occurring Cas9 molecules, e.g., a Cas9 molecule ofS aureus, S. pyogenes, or C. jejuni as follows: its ability to modulate,e.g., decreased or increased, cleavage of a double stranded break(endonuclease and/or exonuclease activity), e.g., as compared to anaturally occurring Cas9 molecule (e.g., a Cas9 molecule of S aureus, S.pyogenes, or C. jejuni); its ability to modulate, e.g., decreased orincreased, cleavage of a single strand of a nucleic acid, e.g., anon-complimentary strand of a nucleic acid molecule or a complementarystrand of a nucleic acid molecule (nickase activity), e.g., as comparedto a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of Saureus, S. pyogenes, or C. jejuni); or the ability to cleave a nucleicacid molecule, e.g., a double stranded or single stranded nucleic acidmolecule, can be eliminated.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is aneaCas9 molecule or eaCas9 polypeptide comprising one or more of thefollowing activities: cleavage activity associated with a RuvC domain;cleavage activity associated with an HNH domain; cleavage activityassociated with an HNH domain and cleavage activity associated with aRuvC domain.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is aneiCas9 molecule or eiCas9 polypeptide which does not cleave a nucleicacid molecule (either double stranded or single stranded nucleic acidmolecules) or cleaves a nucleic acid molecule with significantly lessefficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavageactivity of a reference Cas9 molecule, e.g., as measured by an assaydescribed herein. The reference Cas9 molecule can be a naturallyoccurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9molecule such as a Cas9 molecule of S. pyogenes, S. thermophilus, S.aureus, C. jejuni or N. meningitidis. In an embodiment, the referenceCas9 molecule is the naturally occurring Cas9 molecule having theclosest sequence identity or homology. In an embodiment, the eiCas9molecule or eiCas9 polypeptide lacks substantial cleavage activityassociated with a RuvC domain and cleavage activity associated with anHNH domain.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is aneaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acidresidues of S. pyogenes shown in the consensus sequence disclosed inFIGS. 2A-2G, and has one or more amino acids that differ from the aminoacid sequence of S. pyogenes (e.g., has a substitution) at one or moreresidue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 aminoacid residues) represented by an “-” in the consensus sequence disclosedin FIGS. 2A-2G or SEQ ID NO: 7.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptidecomprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensussequence disclosed in FIGS. 2A-2G differs at no more than 1, 2, 3, 4, 5,10, 15, or 20% of the fixed residues in the consensus sequence disclosedin FIGS. 2A-2G;

the sequence corresponding to the residues identified by “*” in theconsensus sequence disclosed in FIGS. 2A-2G differ at no more than 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from thecorresponding sequence of naturally occurring Cas9 molecule, e.g., an S.pyogenes Cas9 molecule; and,

the sequence corresponding to the residues identified by “-” in theconsensus sequence disclosed in FIGS. 2A-2G differ at no more than 5,10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “-” residues from thecorresponding sequence of naturally occurring Cas9 molecule, e.g., an S.pyogenes Cas9 molecule.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is aneaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acidresidues of S. thermophilus shown in the consensus sequence disclosed inFIGS. 2A-2G, and has one or more amino acids that differ from the aminoacid sequence of S. thermophilus (e.g., has a substitution) at one ormore residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200amino acid residues) represented by an “-” in the consensus sequencedisclosed in FIGS. 2A-2G.

In an embodiment the altered Cas9 molecule or Cas9 polypeptide comprisesa sequence in which:

the sequence corresponding to the fixed sequence of the consensussequence disclosed in FIGS. 2A-2G differs at no more than 1, 2, 3, 4, 5,10, 15, or 20% of the fixed residues in the consensus sequence disclosedin FIGS. 2A-2G;

the sequence corresponding to the residues identified by “*” in theconsensus sequence disclosed in FIGS. 2A-2G differ at no more than 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from thecorresponding sequence of naturally occurring Cas9 molecule, e.g., an S.thermophilus Cas9 molecule; and,

the sequence corresponding to the residues identified by “-” in theconsensus sequence disclosed in FIGS. 2A-2G differ at no more than 5,10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “-” residues from thecorresponding sequence of naturally occurring Cas9 molecule, e.g., an S.thermophilus Cas9 molecule.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is aneaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acidresidues of S. mutans shown in the consensus sequence disclosed in FIGS.2A-2G, and has one or more amino acids that differ from the amino acidsequence of S. mutans (e.g., has a substitution) at one or more residue(e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acidresidues) represented by an “-” in the consensus sequence disclosed inFIGS. 2A-2G.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptidecomprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensussequence disclosed in FIGS. 2A-2G differs at no more than 1, 2, 3, 4, 5,10, 15, or 20% of the fixed residues in the consensus sequence disclosedin FIGS. 2A-2G;

the sequence corresponding to the residues identified by “*” in theconsensus sequence disclosed in FIGS. 2A-2G differ at no more than 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from thecorresponding sequence of naturally occurring Cas9 molecule, e.g., an S.mutans Cas9 molecule; and,

the sequence corresponding to the residues identified by “-” in theconsensus sequence disclosed in FIGS. 2A-2G differ at no more than 5,10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “-” residues from thecorresponding sequence of naturally occurring Cas9 molecule, e.g., an S.mutans Cas9 molecule.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide is aneaCas9 molecule or eaCas9 polypeptide comprising the fixed amino acidresidues of L. innocula shown in the consensus sequence disclosed inFIGS. 2A-2G, and has one or more amino acids that differ from the aminoacid sequence of L. innocula (e.g., has a substitution) at one or moreresidue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 aminoacid residues) represented by an “-” in the consensus sequence disclosedin FIGS. 2A-2G.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptidecomprises a sequence in which:

the sequence corresponding to the fixed sequence of the consensussequence disclosed in FIGS. 2A-2G differs at no more than 1, 2, 3, 4, 5,10, 15, or 20% of the fixed residues in the consensus sequence disclosedin FIGS. 2A-2G;

the sequence corresponding to the residues identified by “*” in theconsensus sequence disclosed in FIGS. 2A-2G differ at no more than 1, 2,3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the “*” residues from thecorresponding sequence of naturally occurring Cas9 molecule, e.g., an L.innocula Cas9 molecule; and,

the sequence corresponding to the residues identified by “-” in theconsensus sequence disclosed in FIGS. 2A-2G differ at no more than 5,10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the “-” residues from thecorresponding sequence of naturally occurring Cas9 molecule, e.g., an L.innocula Cas9 molecule.

In an embodiment, the altered Cas9 molecule or Cas9 polypeptide, e.g.,an eaCas9 molecule, can be a fusion, e.g., of two of more different Cas9molecules or Cas9 polypeptides, e.g., of two or more naturally occurringCas9 molecules of different species. For example, a fragment of anaturally occurring Cas9 molecule of one species can be fused to afragment of a Cas9 molecule of a second species. As an example, afragment of Cas9 molecule of S. pyogenes comprising an N-terminalRuvC-like domain can be fused to a fragment of Cas9 molecule of aspecies other than S. pyogenes (e.g., S. thermophilus) comprising anHNH-like domain.

Cas9 Molecules and Cas9 Polypeptides with Altered PAM Recognition or NoPAM Recognition

Naturally occurring Cas9 molecules can recognize specific PAM sequences,for example, the PAM recognition sequences described above for S.pyogenes, S. thermophilus, S. mutans, S. aureus and N. meningitidis.

In an embodiment, a Cas9 molecule or Cas9 polypeptide has the same PAMspecificities as a naturally occurring Cas9 molecule. In otherembodiments, a Cas9 molecule or Cas9 polypeptide has a PAM specificitynot associated with a naturally occurring Cas9 molecule, or a PAMspecificity not associated with the naturally occurring Cas9 molecule towhich it has the closest sequence homology. For example, a naturallyoccurring Cas9 molecule can be altered, e.g., to alter PAM recognition,e.g., to alter the PAM sequence that the Cas9 molecule recognizes todecrease off target sites and/or improve specificity; or eliminate a PAMrecognition requirement. In an embodiment, a Cas9 molecule or Cas9polypeptide can be altered, e.g., to increase length of PAM recognitionsequence and/or improve Cas9 specificity to high level of identity,e.g., to decrease off target sites and increase specificity. In anembodiment, the length of the PAM recognition sequence is at least 4, 5,6, 7, 8, 9, 10 or 15 amino acids in length. Cas9 molecules or Cas9polypeptides that recognize different PAM sequences and/or have reducedoff-target activity can be generated using directed evolution. Exemplarymethods and systems that can be used for directed evolution of Cas9molecules are described, e.g., in Esvelt 2011. Candidate Cas9 moleculescan be evaluated, e.g., by methods described in Section V.

Alterations of the PI domain, which mediates PAM recognition, arediscussed below.

Synthetic Cas9 Molecules and Cas9 Polypeptides with Altered PI Domains

Current genome-editing methods are limited in the diversity of targetsequences that can be targeted by the PAM sequence that is recognized bythe Cas9 molecule utilized. A synthetic Cas9 molecule (or Syn-Cas9molecule), or synthetic Cas9 polypeptide (or Syn-Cas9 polypeptide), asthat term is used herein, refers to a Cas9 molecule or Cas9 polypeptidethat comprises a Cas9 core domain from one bacterial species and afunctional altered PI domain, i.e., a PI domain other than thatnaturally associated with the Cas9 core domain, e.g., from a differentbacterial species.

In an embodiment, the altered PI domain recognizes a PAM sequence thatis different from the PAM sequence recognized by the naturally-occurringCas9 from which the Cas9 core domain is derived. In an embodiment, thealtered PI domain recognizes the same PAM sequence recognized by thenaturally-occurring Cas9 from which the Cas9 core domain is derived, butwith different affinity or specificity. A Syn-Cas9 molecule or Syn-Cas9polypeptide can be, respectively, a Syn-eaCas9 molecule or Syn-eaCas9polypeptide or a Syn-eiCas9 molecule Syn-eiCas9 polypeptide.

An exemplary Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises:

a) a Cas9 core domain, e.g., a Cas9 core domain from Table 12 or 13,e.g., a S. aureus, S. pyogenes, or C. jejuni Cas9 core domain; and

b) an altered PI domain from a species X Cas9 sequence selected fromTables 15 and 16.

In an embodiment, the RKR motif (the PAM binding motif) of said alteredPI domain comprises: differences at 1, 2, or 3 amino acid residues; adifference in amino acid sequence at the first, second, or thirdposition; differences in amino acid sequence at the first and secondpositions, the first and third positions, or the second and thirdpositions; as compared with the sequence of the RKR motif of the nativeor endogenous PI domain associated with the Cas9 core domain.

In an embodiment, the Cas9 core domain comprises the Cas9 core domainfrom a species X Cas9 from Table 12 and said altered PI domain comprisesa PI domain from a species Y Cas9 from Table 12.

In an embodiment, the RKR motif of the species X Cas9 is other than theRKR motif of the species Y Cas9.

In an embodiment, the RKR motif of the altered PI domain is selectedfrom XXY, XNG, and XNQ.

In an embodiment, the altered PI domain has at least 60, 70, 80, 90, 95,or 100% homology with the amino acid sequence of a naturally occurringPI domain of said species Y from Table 12.

In an embodiment, the altered PI domain differs by no more than 50, 40,30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residue from the aminoacid sequence of a naturally occurring PI domain of said second speciesfrom Table 12.

In an embodiment, the Cas9 core domain comprises a S. aureus core domainand altered PI domain comprises: an A. denitrificans PI domain; a C.jejuni PI domain; a H. mustelae PI domain; or an altered PI domain ofspecies X PI domain, wherein species X is selected from Table 16.

In an embodiment, the Cas9 core domain comprises a S. pyogenes coredomain and the altered PI domain comprises: an A. denitrificans PIdomain; a C. jejuni PI domain; a H. mustelae PI domain; or an altered PIdomain of species X PI domain, wherein species X is selected from Table16.

In an embodiment, the Cas9 core domain comprises a C. jejuni core domainand the altered PI domain comprises: an A. denitrificans PI domain; a H.mustelae PI domain; or an altered PI domain of species X PI domain,wherein species X is selected from Table 16.

In an embodiment, the Cas9 molecule or Cas9 polypeptide furthercomprises a linker disposed between said Cas9 core domain and saidaltered PI domain.

In an embodiment, the linker comprises: a linker described elsewhereherein disposed between the Cas9 core domain and the heterologous PIdomain. Suitable linkers are further described in Section VI.

Exemplary altered PI domains for use in Syn-Cas9 molecules are describedin Tables 15 and 16. The sequences for the 83 Cas9 orthologs referencedin Tables 15 and 16 are provided in Table 12. Table 14 provides the Cas9orthologs with known PAM sequences and the corresponding RKR motif.

In an embodiment, a Syn-Cas9 molecule or Syn-Cas9 polypeptide may alsobe size-optimized, e.g., the Syn-Cas9 molecule or Syn-Cas9 polypeptidecomprises one or more deletions, and optionally one or more linkersdisposed between the amino acid residues flanking the deletions. In anembodiment, a Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises a RECdeletion.

Size-Optimized Cas9 Molecules and Cas9 Polypeptides

Engineered Cas9 molecules and engineered Cas9 polypeptides describedherein include a Cas9 molecule or Cas9 polypeptide comprising a deletionthat reduces the size of the molecule while still retaining desired Cas9properties, e.g., essentially native conformation, Cas9 nucleaseactivity, and/or target nucleic acid molecule recognition. Providedherein are Cas9 molecules or Cas9 polypeptides comprising one or moredeletions and optionally one or more linkers, wherein a linker isdisposed between the amino acid residues that flank the deletion.Methods for identifying suitable deletions in a reference Cas9 molecule,methods for generating Cas9 molecules with a deletion and a linker, andmethods for using such Cas9 molecules will be apparent to one ofordinary skill in the art upon review of this document.

A Cas9 molecule, e.g., a S. aureus, S. pyogenes, or C. jejuni, Cas9molecule, having a deletion is smaller, e.g., has reduced number ofamino acids, than the corresponding naturally-occurring Cas9 molecule.The smaller size of the Cas9 molecules allows increased flexibility fordelivery methods, and thereby increases utility for genome-editing. ACas9 molecule or Cas9 polypeptide can comprise one or more deletionsthat do not substantially affect or decrease the activity of theresultant Cas9 molecules or Cas9 polypeptides described herein.Activities that are retained in the Cas9 molecules or Cas9 polypeptidescomprising a deletion as described herein include one or more of thefollowing:

a nickase activity, i.e., the ability to cleave a single strand, e.g.,the non-complementary strand or the complementary strand, of a nucleicacid molecule; a double stranded nuclease activity, i.e., the ability tocleave both strands of a double stranded nucleic acid and create adouble stranded break, which in an embodiment is the presence of twonickase activities;

an endonuclease activity;

an exonuclease activity;

a helicase activity, i.e., the ability to unwind the helical structureof a double stranded nucleic acid;

and recognition activity of a nucleic acid molecule, e.g., a targetnucleic acid or a gRNA.

Activity of the Cas9 molecules or Cas9 polypeptides described herein canbe assessed using the activity assays described herein or in the art.

Identifying Regions Suitable for Deletion

Suitable regions of Cas9 molecules for deletion can be identified by avariety of methods. Naturally-occurring orthologous Cas9 molecules fromvarious bacterial species, e.g., any one of those listed in Table 12,can be modeled onto the crystal structure of S. pyogenes Cas9 (Nishimasu2014) to examine the level of conservation across the selected Cas9orthologs with respect to the three-dimensional conformation of theprotein. Less conserved or unconserved regions that are spatiallylocated distant from regions involved in Cas9 activity, e.g., interfacewith the target nucleic acid molecule and/or gRNA, represent regions ordomains are candidates for deletion without substantially affecting ordecreasing Cas9 activity.

REC-Optimized Cas9 Molecules and Cas9 Polypeptides

A REC-optimized Cas9 molecule, or a REC-optimized Cas9 polypeptide, asthat term is used herein, refers to a Cas9 molecule or Cas9 polypeptidethat comprises a deletion in one or both of the REC2 domain and theRE1_(CT) domain (collectively a REC deletion), wherein the deletioncomprises at least 10% of the amino acid residues in the cognate domain.A REC-optimized Cas9 molecule or Cas9 polypeptide can be an eaCas9molecule or eaCas9 polypeptide, or an eiCas9 molecule or eiCas9polypeptide. An exemplary REC-optimized Cas9 molecule or REC-optimizedCas9 polypeptide comprises:

a) a deletion selected from:

-   -   i) a REC2 deletion;    -   ii) a REC1_(CT) deletion; or    -   iii) a REC1_(SUB) deletion.

Optionally, a linker is disposed between the amino acid residues thatflank the deletion.

In an embodiment, a Cas9 molecule or Cas9 polypeptide includes only onedeletion, or only two deletions. A Cas9 molecule or Cas9 polypeptide cancomprise a REC2 deletion and a REC1_(CT) deletion. A Cas9 molecule orCas9 polypeptide can comprise a REC2 deletion and a REC1_(SUB) deletion.

Generally, the deletion will contain at least 10% of the amino acids inthe cognate domain, e.g., a REC2 deletion will include at least 10% ofthe amino acids in the REC2 domain.

A deletion can comprise: at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%of the amino acid residues of its cognate domain; all of the amino acidresidues of its cognate domain; an amino acid residue outside itscognate domain; a plurality of amino acid residues outside its cognatedomain; the amino acid residue immediately N terminal to its cognatedomain; the amino acid residue immediately C terminal to its cognatedomain; the amino acid residue immediately N terminal to its cognate andthe amino acid residue immediately C terminal to its cognate domain; aplurality of, e.g., up to 5, 10, 15, or 20, amino acid residues Nterminal to its cognate domain; a plurality of, e.g., up to 5, 10, 15,or 20, amino acid residues C terminal to its cognate domain; a pluralityof, e.g., up to 5, 10, 15, or 20, amino acid residues N terminal to itscognate domain and a plurality of e.g., up to 5, 10, 15, or 20, aminoacid residues C terminal to its cognate domain.

In an embodiment, a deletion does not extend beyond: its cognate domain;the N terminal amino acid residue of its cognate domain; the C terminalamino acid residue of its cognate domain.

A REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide caninclude a linker disposed between the amino acid residues that flank thedeletion. Suitable linkers for use between the amino acid resides thatflank a REC deletion in a REC-optimized Cas9 molecule or REC-optimizedCas9 polypeptide is disclosed in Section VI.

In an embodiment, a REC-optimized Cas9 molecule or REC-optimized Cas9polypeptide comprises an amino acid sequence that, other than any RECdeletion and associated linker, has at least 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 99, or 100% homology with the amino acid sequence of anaturally occurring Cas 9, e.g., a Cas9 molecule described in Table 12,e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9 molecule, or a C.jejuni Cas9 molecule.

In an embodiment, a REC-optimized Cas9 molecule or REC-optimized Cas9polypeptide comprises an amino acid sequence that, other than any RECdeletion and associated linker, differs by no more than 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, or 25, amino acid residues from the amino acidsequence of a naturally occurring Cas 9, e.g., a Cas9 molecule describedin Table 12, e.g., a S. aureus Cas9 molecule, a S. pyogenes Cas9molecule, or a C. jejuni Cas9 molecule.

In an embodiment, a REC-optimized Cas9 molecule or REC-optimized Cas9polypeptide comprises an amino acid sequence that, other than any RECdeletion and associate linker, differs by no more than 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, or 25% of the, amino acid residues from the aminoacid sequence of a naturally occurring Cas 9, e.g., a Cas9 moleculedescribed in Table 12, e.g., a S. aureus Cas9 molecule, a S. pyogenesCas9 molecule, or a C. jejuni Cas9 molecule.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. Methods of alignment of sequences forcomparison are well known in the art. Optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman 1981, by the homology alignment algorithm of Needleman& Wunsch 1970, by the search for similarity method of Pearson & Lipman1988, by computerized implementations of these algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package, GeneticsComputer Group, 575 Science Dr., Madison, Wis.), or by manual alignmentand visual inspection (see, e.g., Brent et al., Current Protocols inMolecular Biology, John Wiley & Sons, Inc. (2003)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul 1977 and Altschul 1990),respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of Myers 1988, which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4. Inaddition, the percent identity between two amino acid sequences can bedetermined using the Needleman & Wunsch 1970 algorithm, which has beenincorporated into the GAP program in the GCG software package (availableat www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6.

Sequence information for exemplary REC deletions are provided for 83naturally-occurring Cas9 orthologs in Table 12. The amino acid sequencesof exemplary Cas9 molecules from different bacterial species are shownbelow.

TABLE 12 Amino Acid Sequence of Cas9 Orthologs REC2 REC1_(CT) Rec_(sub)start stop # AA start stop # AA start stop # AA Amino acid (AA (AAdeleted (AA (AA deleted (AA (AA deleted Species/Composite ID sequencepos) pos) (n) pos) pos) (n) pos) pos) (n) Staphylococcus aureus SEQ IDNO: 126 166 41 296 352 57 296 352 57 tr|J7RUA5|J7RUA5_STA 26 AUStreptococcus pyogenes SEQ ID NO: 176 314 139 511 592 82 511 592 82sp|Q99ZW2|CAS9_STRP 2 1 Campylobacter jejuni SEQ ID NO: 137 181 45 316360 45 316 360 45 NCTC 11168 306 gi|218563121|ref|YP_002 344900.1Bacteroides fragilis SEQ ID NO: 148 339 192 524 617 84 524 617 84 NCTC9343 307 gi|60683389|ref|YP_2135 33.1| Bifidobacterium bifidum SEQ IDNO: 173 335 163 516 607 87 516 607 87 S17 308 gi|310286728|ref|YP_003937986. Veillonella atypica ACS- SEQ ID NO: 185 339 155 574 663 79 574663 79 134-V-Col7a 309 gi|303229466|ref|ZP_073 16256.1 Lactobacillusrhamnosus SEQ ID NO: 169 320 152 559 645 78 559 645 78 GG 310gi|258509199|ref|YP_003 171950.1 Filifactor alocis ATCC SEQ ID NO: 166314 149 508 592 76 508 592 76 35896 311 gi|374307738|ref|YP_005 054169.1Oenococcus kitaharae SEQ ID NO: 169 317 149 555 639 80 555 639 80 DSM17330 312 gi|366983953|gb|EHN593 52.1| Fructobacillus fructosus SEQ IDNO: 168 314 147 488 571 76 488 571 76 KCTC 3544 313gi|339625081|ref|ZP_086 60870.1 Catenibacterium SEQ ID NO: 173 318 146511 594 78 511 594 78 mitsuokai DSM 15897 314 gi|224543312|ref|ZP_03683851.1 Finegoldia magna ATCC SEQ ID NO: 168 313 146 452 534 77 452 53477 29328 315 gi|169823755|ref|YP_001 691366.1 Coriobacterium SEQ ID NO:175 318 144 511 592 82 511 592 82 glomerans PW2 316gi|328956315|ref|YP_004 373648.1 Eubacterium yurii ATCC SEQ ID NO: 169310 142 552 633 76 552 633 76 43715 317 gi|306821691|ref|ZP_074 55288.1Peptoniphilus duerdenii SEQ ID NO: 171 311 141 535 615 76 535 615 76ATCC BAA-1640 318 gi|304438954|ref|ZP_073 98877.1 Acidaminococcus sp.D21 SEQ ID NO: 167 306 140 511 591 75 511 591 75 gi|227824983|ref|ZP_039319 89815.1 Lactobacillus farciminis SEQ ID NO: 171 310 140 542 621 85542 621 85 KCTC 3681 320 gi|336394882|ref|ZP_085 76281.1 Streptococcussanguinis SEQ ID NO: 185 324 140 411 490 85 411 490 85 SK49 321gi|422884106|ref|ZP_169 30555.1 Coprococcus catus GD-7 SEQ ID NO: 172310 139 556 634 76 556 634 76 gi|291520705|emb|CBK7 322 8998.1|Streptococcus mutans SEQ ID NO: 176 314 139 392 470 84 392 470 84 UA1591 gi|24379809|ref|NP_7217 64.1| Streptococcus pyogenes SEQ ID NO: 176314 139 523 600 82 523 600 82 M1 GAS 2 gi|13622193|gb|AAK339 36.1|Streptococcus SEQ ID NO: 176 314 139 481 558 81 481 558 81 thermophilusLMD-9 3 gi|116628213|ref|YP_820 832.1| Fusobacterium nucleatum SEQ IDNO: 171 308 138 537 614 76 537 614 76 ATCC 49256 326gi|34762592|ref|ZP_0014 3587.1| Planococcus antarcticus SEQ ID NO: 162299 138 538 614 94 538 614 94 DSM 14505 327 gi|389815359|ref|ZP_10206685.1 Treponema denticola SEQ ID NO: 169 305 137 524 600 81 524 600 81ATCC 35405 328 gi|42525843|ref|NP_9709 41.1| Solobacterium moorei SEQ IDNO: 179 314 136 544 619 77 544 619 77 F0204 329 gi|320528778|ref|ZP_08029929.1 Staphylococcus SEQ ID NO: 164 299 136 531 606 92 531 606 92pseudintermedius ED99 330 gi|323463801|gb|ADX75 954.1| FlavobacteriumSEQ ID NO: 162 286 125 538 613 63 538 613 63 branchiophilum FL-15 331gi|347536497|ref|YP_004 843922.1 Ignavibacterium album SEQ ID NO: 223329 107 357 432 90 357 432 90 JCM 16511 332 gi|385811609|ref|YP_005848005.1 Bergeyella zoohelcum SEQ ID NO: 165 261 97 529 604 56 529 60456 ATCC 43767 333 gi|423317190|ref|ZP_172 95095.1 Nitrobacterhamburgensis SEQ ID NO: 169 253 85 536 611 48 536 611 48 X14 334gi|92109262|ref|YP_5715 50.1| Odoribacter laneus YIT SEQ ID NO: 164 24279 535 610 63 535 610 63 12061 335 gi|374384763|ref|ZP_096 42280.1Legionella pneumophila SEQ ID NO: 164 239 76 402 476 67 402 476 67 str.Paris 336 gi|54296138|ref|YP_1225 07.1| Bacteroides sp. 20_3 SEQ ID NO:198 269 72 530 604 83 530 604 83 gi|301311869|ref|ZP_072 337 17791.1Akkermansia muciniphila SEQ ID NO: 136 202 67 348 418 62 348 418 62 ATCCBAA-835 338 gi|187736489|ref|YP_001 878601. Prevotella sp. C561 SEQ IDNO: 184 250 67 357 425 78 357 425 78 gi|345885718|ref|ZP_088 339 37074.1Wolinella succinogenes SEQ ID NO: 157 218 36 401 468 60 401 468 60 DSM1740 340 gi|34557932|ref|NP_9077 47.1| Alicyclobacillus SEQ ID NO: 142196 55 416 482 61 416 482 61 hesperidum URH17-3-68 341gi|403744858|ref|ZP_109 53934.1 Caenispirillum salinarum SEQ ID NO: 161214 54 330 393 68 330 393 68 AK4 342 gi|427429481|ref|ZP_189 19511.1Eubacterium rectale SEQ ID NO: 133 185 53 322 384 60 322 384 60 ATCC33656 343 gi|238924075|ref|YP_002 937591.1 Mycoplasma synoviae 53 SEQ IDNO: 187 239 53 319 381 80 319 381 80 gi|71894592|ref|YP_2787 344 00.1|Porphyromonas sp. oral SEQ ID NO: 150 202 53 309 371 60 309 371 60 taxon279 str. F0450 345 gi|402847315|ref|ZP_108 95610.1 Streptococcus SEQ IDNO: 127 178 139 424 486 81 424 486 81 thermophilus LMD-9 346gi|116627542|ref|YP_820 161.1| Roseburia inulinivorans SEQ ID NO: 154204 51 318 380 69 318 380 69 DSM 16841 347 gi|225377804|ref|ZP_03755025.1 Methylosinus SEQ ID NO: 144 193 50 426 488 64 426 488 64trichosporium OB3b 348 gi|296446027|ref|ZP_068 87976.1 Ruminococcusalbus 8 SEQ ID NO: 139 187 49 351 412 55 351 412 55gi|325677756|ref|ZP_081 349 57403.1 Bifidobacterium longum SEQ ID NO:183 230 48 370 431 44 370 431 44 DJO10A 350 gi|189440764|ref|YP_001955845. Enterococcus faecalis SEQ ID NO: 123 170 48 327 387 60 327 38760 TX0012 351 gi|315149830|gb|EFT938 46.1| Mycoplasma mobile SEQ ID NO:179 226 48 314 374 79 314 374 79 163K 352 gi|47458868|ref|YP_0157 30.1|Actinomyces coleocanis SEQ ID NO: 147 193 47 358 418 40 358 418 40 DSM15436 353 gi|227494853|ref|ZP_039 25169.1 Dinoroseobacter shibae SEQ IDNO: 138 184 47 338 398 48 338 398 48 DFL 12 354 gi|159042956|ref|YP_001531750.1 Actinomyces sp. oral SEQ ID NO: 183 228 46 349 409 40 349 40940 taxon 180 str. F0310 355 gi|315605738|ref|ZP_078 80770.1 Alcanivoraxsp. W11-5 SEQ ID NO: 139 183 45 344 404 61 344 404 61gi|407803669|ref|ZP_111 356 50502.1 Aminomonas paucivorans SEQ ID NO:134 178 45 341 401 63 341 401 63 DSM 12260 357 gi|312879015|ref|ZP_07738815.1 Mycoplasma canis PG 14 SEQ ID NO: 139 183 45 319 379 76 319 37976 gi|384393286|gb|EIE3973 358 6.1| Lactobacillus SEQ ID NO: 141 184 44328 387 61 328 387 61 coryniformis KCTC 3535 359 gi|336393381|ref|ZP_08574780.1 Elusimicrobium minutum SEQ ID NO: 177 219 43 322 381 47 322 38147 Pei191 360 gi|187250660|ref|YP_001 875142.1 Neisseria meningitidisSEQ ID NO: 147 189 43 360 419 61 360 419 61 Z2491 25gi|218767588|ref|YP_002 342100.1 Pasteurella multocida str. SEQ ID NO:139 181 43 319 378 61 319 378 61 Pm70 362 gi|15602992|ref|NP_2460 64.1|Rhodovulum sp. PH10 SEQ ID NO: 141 183 43 319 378 48 319 378 48gi|402849997|ref|ZP_108 363 98214.1 Eubacterium dolichum SEQ ID NO: 131172 42 303 361 59 303 361 59 DSM 3991 364 gi|160915782|ref|ZP_02077990.1 Nitratifractor salsuginis SEQ ID NO: 143 184 42 347 404 61 347404 61 DSM 16511 365 gi|319957206|ref|YP_004 168469.1 Rhodospirillumrubrum SEQ ID NO: 139 180 42 314 371 55 314 371 55 ATCC 11170 366gi|83591793|ref|YP_4255 45.1| Clostridium SEQ ID NO: 137 176 40 320 37661 320 376 61 cellulolyticum H10 367 gi|220930482|ref|YP_002 507391.1Helicobacter mustelae SEQ ID NO: 148 187 40 298 354 48 298 354 48 12198368 gi|291276265|ref|YP_003 516037.1 Ilyobacter polytropus SEQ ID NO:134 173 40 462 517 63 462 517 63 DSM 2926 369 gi|310780384|ref|YP_003968716.1 Sphaerochaeta globus str. SEQ ID NO: 163 202 40 335 389 45 335389 45 Buddy 370 gi|325972003|ref|YP_004 248194.1 Staphylococcus SEQ IDNO: 128 167 40 337 391 57 337 391 57 lugdunensis M23590 371gi|315659848|ref|ZP_079 12707.1 Treponema sp. JC4 SEQ ID NO: 144 183 40328 382 63 328 382 63 gi|384109266|ref|ZP_100 372 10146.1 Uncultured SEQID NO: 154 193 40 313 365 55 313 365 55 Deltaproteobacterium 373 HF007007E19 gi|297182908|gb|ADI190 58.1| Alicycliphilus SEQ ID NO: 140 178 39317 366 48 317 366 48 denitrificans K601 374 gi|330822845|ref|YP_004386148.1 Azospirillum sp. B510 SEQ ID NO: 205 243 39 342 389 46 342 38946 gi|288957741|ref|YP_003 375 448082.1 Bradyrhizobium sp. SEQ ID NO:143 181 39 323 370 48 323 370 48 BTAi1 376 gi|148255343|ref|YP_001239928.1 Parvibaculum SEQ ID NO: 138 176 39 327 374 58 327 374 58lavamentivorans DS-1 377 gi|154250555|ref|YP_001 411379.1 Prevotellatimonensis SEQ ID NO: 170 208 39 328 375 61 328 375 61 CRIS 5C-B1 378gi|282880052|ref|ZP_062 88774.1 Bacillus smithii 7 3 SEQ ID NO: 134 17138 401 448 63 401 448 63 47FAA 379 gi|365156657|ref|ZP_093 52959.1Candidatus SEQ ID NO: 135 172 38 344 391 53 344 391 53 Puniceispirillummarinum 380 IMCC1322 gi|294086111|ref|YP_003 552871.1 Barnesiella SEQ IDNO: 140 176 37 371 417 60 371 417 60 intestinihominis YIT 381 11860gi|404487228|ref|ZP_110 22414.1 Ralstonia syzygii R24 SEQ ID NO: 140 17637 395 440 50 395 440 50 gi|344171927|emb|CCA8 382 4553.1| Wolinellasuccinogenes SEQ ID NO: 145 180 36 348 392 60 348 392 60 DSM 1740 383gi|34557790|ref|NP_9076 05.1| Mycoplasma SEQ ID NO: 144 177 34 373 41671 373 416 71 gallisepticum str. F 384 gi|284931710|gb|ADC316 48.1|Acidothermus SEQ ID NO: 150 182 33 341 380 58 341 380 58 cellulolyticus11B 385 gi|117929158|ref|YP_873 709.1| Mycoplasma SEQ ID NO: 156 184 29381 420 62 381 420 62 ovipneumoniae SC01 386 gi|363542550|ref|ZP_09312133.1

TABLE 13 Amino Acid Sequence of Cas9 Core Domains Cas9 Start Cas9 Stop(AA pos) (AA pos) Start and Stop numbers refer to the Strain Namesequence in Table 11 Staphylococcus 1 772 aureus Streptococcus 1 1099pyogenes Campulobacter jejuni 1 741

TABLE 14 Identified PAM sequences and corresponding RKR motifs. PAM RKRsequence motif Strain Name (NA) (AA) Streptococcus pyogenes NGG RKRStreptococcus mutans NGG RKR Streptococcus thermophilus A NGGNG RYRTreponema denticola NAAAAN VAK Streptococcus thermophilus B NNAAAAW IYKCampylobacter jejuni NNNNACA NLK Pasteurella multocida GNNNCNNA KDGNeisseria meningitidis NNNNGATT or IGK Staphylococcus aureus NNGRRV NDK(R = A or G; V = A. G or C) NNGRRT (R = A or G)PI domains are provided in Tables 15 and 16.

TABLE 15 Altered PI Domains PI Start PI Stop (AA pos) (AA pos) Start andStop numbers refer to the RKR sequences in Table Length of motif StrainName 11 PI (AA) (AA) Alicycliphilus denitrificans 837 1029 193 --Y K601Campylobacter jejuni 741 984 244 -NG NCTC 11168 Helicobacter mustelae771 1024 254 -NQ 12198

TABLE 16 Other Altered PI Domains PI Start PI Stop (AA pos) (AA pos)Start and Stop numbers refer to Length RKR the sequences in of motifStrain Name Table 11 PI (AA) (AA) Akkermansia muciniphila ATCC 871 1101231 ALK BAA-835 Ralstonia syzygii R24 821 1062 242 APY Cand.Puniceispirillum marinum 815 1035 221 AYK IMCC1322 Fructobacillusfructosus 1074 1323 250 DGN KCTC 3544 Eubacterium yurii ATCC 43715 11071391 285 DGY Eubacterium dolichum DSM 3991 779 1096 318 DKKDinoroseobacter shibae DFL 12 851 1079 229 DPI Clostridiumcellulolyticum H10 767 1021 255 EGK Pasteurella multocida str. Pm70 8151056 242 ENN Mycoplasma canis PG 14 907 1233 327 EPK Porphyromonas sp.oral taxon 279 935 1197 263 EPT str. F0450 Filifactor alocis ATCC 358961094 1365 272 EVD Aminomonas paucivorans DSM 801 1052 252 EVY 12260Wolinella succinogenes DSM 1740 1034 1409 376 EYK Oenococcus kitaharaeDSM 17330 1119 1389 271 GAL CoriobacteriumglomeransPW2 1126 1384 259 GDRPeptoniphilus duerdenii ATCC 1091 1364 274 GDS BAA-1640 Bifidobacteriumbifidum S17 1138 1420 283 GGL Alicyclobacillus hesperidum 876 1146 271GGR URH17-3-68 Roseburia inulinivorans DSM 895 1152 258 GGT 16841Actinomyces coleocanis DSM 843 1105 263 GKK 15436 Odoribacter laneus YIT12061 1103 1498 396 GKV Coprococcus catus GD-7 1063 1338 276 GNQEnterococcus faecalis TX0012 829 1150 322 GRK Bacillus smithii 7 3 47FAA809 1088 280 GSK Legionella pneumophila str. Paris 1021 1372 352 GTMBacteroides fragilis NCTC 9343 1140 1436 297 IPV Mycoplasmaovipneumoniae SC01 923 1265 343 IRI Actinomyces sp. oral taxon 180 str.895 1181 287 KEK F0310 Treponema sp. JC4 832 1062 231 KISFusobacteriumnucleatum 1073 1374 302 KKV ATCC49256 Lactobacillusfarciminis KCTC 1101 1356 256 KKV 3681 Nitratifractor salsuginis 8401132 293 KMR DSM 16511 Lactobacillus coryniformis KCTC 850 1119 270 KNK3535 Mycoplasma mobile 163K 916 1236 321 KNY Flavobacteriumbranchiophilum 1182 1473 292 KQK FL-15 Prevotella timonensis CRIS 5C-B1957 1218 262 KQQ Methylosinus trichosporium OB3b 830 1082 253 KRPPrevotella sp. C561 1099 1424 326 KRY Mycoplasma gallisepticum str. F911 1269 359 KTA Lactobacillus rhamnosus GG 1077 1363 287 KYG Wolinellasuccinogenes DSM 1740 811 1059 249 LPN Streptococcus thermophilus LMD-91099 1388 290 MLA Treponema denticola ATCC 35405 1092 1395 304 NDSBergeyella zoohelcum ATCC 43767 1098 1415 318 NEK Veillonella atypicaACS-134-V- 1107 1398 292 NGF Col7a Neisseria meningitidis Z2491 835 1082248 NHN Ignavibacterium album JCM 16511 1296 1688 393 NKK Ruminococcusalbus 8 853 1156 304 NNF Streptococcus thermophilus LMD-9 811 1121 311NNK Barnesiella intestinihominis YIT 871 1153 283 NPV 11860 Azospirillumsp. B510 911 1168 258 PFH Rhodospirillum rubrum ATCC 863 1173 311 PRG11170 Planococcus antarcticus DSM 1087 1333 247 PYY 14505 Staphylococcus1073 1334 262 QIV pseudintermedius ED99 Alcanivorax sp. W11-5 843 1113271 RIE Bradyrhizobium sp. BTAil 811 1064 254 RIY Streptococcus pyogenesM1 GAS 1099 1368 270 RKR Streptococcus mutans UA159 1078 1345 268 RKRStreptococcus Pyogenes 1099 1368 270 RKR Bacteroides sp. 20 3 1147 1517371 RNI S. aureus 772 1053 282 RNK Solobacterium moorei F0204 1062 1327266 RSG Finegoldia magna ATCC 29328 1081 1348 268 RTE uncultured deltaproteobacterium 770 1011 242 SGG HF0070 07E19 Acidaminococcus sp. D211064 1358 295 SIG Eubacterium rectale ATCC 33656 824 1114 291 SKKCaenispirillum salinarum AK4 1048 1442 395 SLV Acidothermuscellulolyticus 11B 830 1138 309 SPS Catenibacterium mitsuokai DSM 10681329 262 SPT 15897 Parvibaculum lavamentivorans 827 1037 211 TGN DS-1Staphylococcus lugdunensis 772 1054 283 TKK M23590 Streptococcussanguinis SK49 1123 1421 299 TRM Elusimicrobium minutum Pei191 910 1195286 TTG Nitrobacter hamburgensis X14 914 1166 253 VAY Mycoplasmasynoviae 53 991 1314 324 VGF Sphaerochaeta globus str. Buddy 877 1179303 VKG Ilyobacter polytropus DSM 2926 837 1092 256 VNG Rhodovulum sp.PH10 821 1059 239 VPY Bifidobacterium longum DJO10A 904 1187 284 VRKNucleic Acids Encoding Cas9 Molecules

Nucleic acids encoding the Cas9 molecules or Cas9 polypeptides, e.g., aneaCas9 molecule or eaCas9 polypeptide, are provided herein.

Exemplary nucleic acids encoding Cas9 molecules or Cas9 polypeptides aredescribed in Cong 2013; Wang 2013; Mali 2013; and Jinek 2012. Anotherexemplary nucleic acid encoding a Cas9 molecule or Cas9 polypeptide isshown in black in FIG. 8.

In an embodiment, a nucleic acid encoding a Cas9 molecule or Cas9polypeptide can be a synthetic nucleic acid sequence. For example, thesynthetic nucleic acid molecule can be chemically modified, e.g., asdescribed in Section VIII. In an embodiment, the Cas9 mRNA has one ormore (e.g., all of the following properties: it is capped,polyadenylated, substituted with 5-methylcytidine and/or pseudouridine.

In addition, or alternatively, the synthetic nucleic acid sequence canbe codon optimized, e.g., at least one non-common codon or less-commoncodon has been replaced by a common codon. For example, the syntheticnucleic acid can direct the synthesis of an optimized messenger mRNA,e.g., optimized for expression in a mammalian expression system, e.g.,described herein.

In addition, or alternatively, a nucleic acid encoding a Cas9 moleculeor Cas9 polypeptide may comprise a nuclear localization sequence (NLS).Nuclear localization sequences are known in the art.

An exemplary codon optimized nucleic acid sequence encoding a Cas9molecule of S. pyogenes is set forth in SEQ ID NO: 22. The correspondingamino acid sequence is set forth in SEQ ID NO: 2.

An exemplary codon optimized nucleic acid sequence encoding a Cas9molecule of N. meningitidis is set forth in SEQ ID NO: 24. Thecorresponding amino acid sequence is set forth in SEQ ID NO: 25.

An amino acid sequence of a S. aureus Cas9 molecule is set forth in SEQID NO: 26. An exemplary codon optimized nucleic acid sequence encoding aCas9 molecule of S. aureus is set forth in SEQ ID NO: 39.

If any of the above Cas9 sequences are fused with a peptide orpolypeptide at the C-terminus, it is understood that the stop codon willbe removed.

Other Cas Molecules and Cas Polypeptides

Various types of Cas molecules or Cas polypeptides can be used topractice the inventions disclosed herein. In some embodiments, Casmolecules of Type II Cas systems are used. In other embodiments, Casmolecules of other Cas systems are used. For example, Type I or Type IIICas molecules may be used. Exemplary Cas molecules (and Cas systems) aredescribed, e.g., in Haft 2005 and Makarova 2011, the contents of bothreferences are incorporated herein by reference in their entirety.Exemplary Cas molecules (and Cas systems) are also shown in Table 17.

TABLE 17 Cas Systems Structure of encoded Families (and System proteinsuperfamily) Gene type or Name from (PDB of encoded name^(‡) subtypeHaft 2005^(§) accessions)^(¶) protein^(#)** Representatives cas1 Type Icas1 3GOD, COG1518 SERP2463, Type II 3LFX and SPy1047 and Type III 2YZSygbT cas2 Type I cas2 2IVY, 2I8E COG1343 and SERP2462, Type II and 3EXCCOG3512 SPy1048, Type III SPy1723 (N- terminal domain) and ygbF cas3′Type I^(‡‡) cas3 NA COG1203 APE1232 and ygcB cas3″ Subtype NA NA COG2254APE1231 and I-A BH0336 Subtype I-B cas4 Subtype cas4 and NA COG1468APE1239 and I-A csa1 BH0340 Subtype I-B Subtype I-C Subtype I-D SubtypeII-B cas5 Subtype cas5a, 3KG4 COG1688 APE1234, I-A cas5d, (RAMP) BH0337,devS Subtype cas5e, and ygcI I-B cas5h, Subtype cas5p, cas5t I-C andcmx5 Subtype I-E cas6 Subtype cas6 and 3I4H COG1583 and PF1131 and I-Acmx6 COG5551 slr7014 Subtype (RAMP) I-B Subtype I-D Subtype III-ASubtype III-B cas6e Subtype cse3 1WJ9 (RAMP) ygcH I-E cas6f Subtype csy42XLJ (RAMP) y1727 I-F cas7 Subtype csa2, csd2, NA COG1857 and devR andygcJ I-A cse4, csh2, COG3649 Subtype csp1 and (RAMP) I-B cst2 SubtypeI-C Subtype I-E cas8a1 Subtype cmx1, cst1, NA BH0338-like LA3191^(§§)and I-A^(‡‡) csx8, csx13 PG2018^(§§) and CXXC- CXXC cas8a2 Subtype csa4and NA PH0918 AF0070, AF1873, I-A^(‡‡) csx9 MJ0385, PF0637, PH0918 andSSO1401 cas8b Subtype csh1 and NA BH0338-like MTH1090 and I-B^(‡‡)TM1802 TM1802 cas8c Subtype csd1 and NA BH0338-like BH0338 I-C^(‡‡) csp2cas9 Type II^(‡‡) csn1 and NA COG3513 FTN_0757 and csx12 SPy1046 cas10Type III^(‡‡) cmr2, csm1 NA COG1353 MTH326, and csx11 Rv2823c^(§§) andTM1794^(§§) cas10d Subtype csc3 NA COG1353 slr7011 I-D^(‡‡) csy1 Subtypecsy1 NA y1724-like y1724 I-F^(‡‡) csy2 Subtype csy2 NA (RAMP) y1725 I-Fcsy3 Subtype csy3 NA (RAMP) y1726 I-F cse1 Subtype cse1 NA YgcL-likeygcL I-E^(‡‡) cse2 Subtype cse2 2ZCA YgcK-like ygcK I-E csc1 Subtypecsc1 NA alr1563-like alr1563 I-D (RAMP) csc2 Subtype csc1 and NA COG1337slr7012 I-D csc2 (RAMP) csa5 Subtype csa5 NA AF1870 AF1870, MJ0380, I-APF0643 and SSO1398 csn2 Subtype csn2 NA SPy1049-like SPy1049 II-A csm2Subtype csm2 NA COG1421 MTH1081 and III-A^(‡‡) SERP2460 csm3 Subtypecsc2 and NA COG1337 MTH1080 and III-A csm3 (RAMP) SERP2459 csm4 Subtypecsm4 NA COG1567 MTH1079 and III-A (RAMP) SERP2458 csm5 Subtype csm5 NACOG1332 MTH1078 and III-A (RAMP) SERP2457 csm6 Subtype APE2256 2WTECOG1517 APE2256 and III-A and csm6 SSO1445 cmr1 Subtype cmr1 NA COG1367PF1130 III-B (RAMP) cmr3 Subtype cmr3 NA COG1769 PF1128 III-B (RAMP)cmr4 Subtype cmr4 NA COG1336 PF1126 III-B (RAMP) cmr5 Subtype cmr5 2ZOPand COG3337 MTH324 and III-B^(‡‡) 2OEB PF1125 cmr6 Subtype cmr6 NACOG1604 PF1124 III-B (RAMP) csb1 Subtype GSU0053 NA (RAMP) Balac_1306and I-U GSU0053 csb2 Subtype NA NA (RAMP) Balac_1305 and I-U^(§§)GSU0054 csb3 Subtype NA NA (RAMP) Balac_1303^(§§) I-U csx17 Subtype NANA NA Btus_2683 I-U csx14 Subtype NA NA NA GSU0052 I-U csx10 Subtypecsx10 NA (RAMP) Caur_2274 I-U csx16 Subtype VVA1548 NA NA VVA1548 III-UcsaX Subtype csaX NA NA SSO1438 III-U csx3 Subtype csx3 NA NA AF1864III-U csx1 Subtype csa3, csx1, 1XMX and COG1517 and MJ1666, III-U csx2,2I71 COG4006 NE0113, PF1127 DXTHG, and TM1812 NE0113 and TIGR02710 csx15Unknown NA NA TTE2665 TTE2665 csf1 Type U csf1 NA NA AFE_1038 csf2 TypeU csf2 NA (RAMP) AFE_1039 csf3 Type U csf3 NA (RAMP) AFE_1040 csf4 TypeU csf4 NA NA AFE_1037V. Functional Analysis of Candidate Molecules

Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9molecule/gRNA molecule complexes, can be evaluated by art-known methodsor as described herein. For example, exemplary methods for evaluatingthe endonuclease activity of Cas9 molecule are described, e.g., in Jinek2012.

Binding and Cleavage Assay: Testing the Endonuclease Activity of Cas9Molecule

The ability of a Cas9 molecule/gRNA molecule complex to bind to andcleave a target nucleic acid can be evaluated in a plasmid cleavageassay. In this assay, synthetic or in vitro-transcribed gRNA molecule ispre-annealed prior to the reaction by heating to 95° C. and slowlycooling down to room temperature. Native or restrictiondigest-linearized plasmid DNA (300 ng (˜8 nM)) is incubated for 60 minat 37° C. with purified Cas9 protein molecule (50-500 nM) and gRNA(50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5,150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl₂. Thereactions are stopped with 5×DNA loading buffer (30% glycerol, 1.2% SDS,250 mM EDTA), resolved by a 0.8 or 1% agarose gel electrophoresis andvisualized by ethidium bromide staining. The resulting cleavage productsindicate whether the Cas9 molecule cleaves both DNA strands, or only oneof the two strands. For example, linear DNA products indicate thecleavage of both DNA strands. Nicked open circular products indicatethat only one of the two strands is cleaved.

Alternatively, the ability of a Cas9 molecule/gRNA molecule complex tobind to and cleave a target nucleic acid can be evaluated in anoligonucleotide DNA cleavage assay. In this assay, DNA oligonucleotides(10 pmol) are radiolabeled by incubating with 5 units T4 polynucleotidekinase and ˜3-6 pmol (˜20-40 mCi) [γ-32P]-ATP in 1×T4 polynucleotidekinase reaction buffer at 37° C. for 30 min, in a 50 μL reaction. Afterheat inactivation (65° C. for 20 min), reactions are purified through acolumn to remove unincorporated label. Duplex substrates (100 nM) aregenerated by annealing labeled oligonucleotides with equimolar amountsof unlabeled complementary oligonucleotide at 95° C. for 3 min, followedby slow cooling to room temperature. For cleavage assays, gRNA moleculesare annealed by heating to 95° C. for 30 s, followed by slow cooling toroom temperature. Cas9 (500 nM final concentration) is pre-incubatedwith the annealed gRNA molecules (500 nM) in cleavage assay buffer (20mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol) in atotal volume of 9 al. Reactions are initiated by the addition of 1 μltarget DNA (10 nM) and incubated for 1 h at 37° C. Reactions arequenched by the addition of 20 μl of loading dye (5 mM EDTA, 0.025% SDS,5% glycerol in formamide) and heated to 95° C. for 5 min. Cleavageproducts are resolved on 12% denaturing polyacrylamide gels containing 7M urea and visualized by phosphorimaging. The resulting cleavageproducts indicate that whether the complementary strand, thenon-complementary strand, or both, are cleaved.

One or both of these assays can be used to evaluate the suitability of acandidate gRNA molecule or candidate Cas9 molecule.

Binding Assay: Testing the Binding of Cas9 Molecule to Target DNA

Exemplary methods for evaluating the binding of Cas9 molecule to targetDNA are described, e.g., in Jinek 2012.

For example, in an electrophoretic mobility shift assay, target DNAduplexes are formed by mixing of each strand (10 nmol) in deionizedwater, heating to 95° C. for 3 min and slow cooling to room temperature.All DNAs are purified on 8% native gels containing 1×TBE. DNA bands arevisualized by UV shadowing, excised, and eluted by soaking gel pieces inDEPC-treated H₂O. Eluted DNA is ethanol precipitated and dissolved inDEPC-treated H₂O. DNA samples are 5′ end labeled with [γ-32P]-ATP usingT4 polynucleotide kinase for 30 min at 37° C. Polynucleotide kinase isheat denatured at 65° C. for 20 min, and unincorporated radiolabel isremoved using a column. Binding assays are performed in buffercontaining 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl₂, 1 mM DTT and 10%glycerol in a total volume of 10 al. Cas9 protein molecule is programmedwith equimolar amounts of pre-annealed gRNA molecule and titrated from100 pM to 1 μM. Radiolabeled DNA is added to a final concentration of 20pM. Samples are incubated for 1 h at 37° C. and resolved at 4° C. on an8% native polyacrylamide gel containing 1×TBE and 5 mM MgCl₂. Gels aredried and DNA visualized by phosphorimaging.

Differential Scanning Flourimetry (DSF)

The thermostability of Cas9-gRNA ribonucleoprotein (RNP) complexes canbe measured via DSF. This technique measures the thermostability of aprotein, which can increase under favorable conditions such as theaddition of a binding RNA molecule, e.g., a gRNA.

The assay is performed using two different protocols, one to test thebest stoichiometric ratio of gRNA:Cas9 protein and another to determinethe best solution conditions for RNP formation.

To determine the best solution to form RNP complexes, a 2 uM solution ofCas9 in water+10× SYPRO Orange® (Life Technologies cat #S-6650) anddispensed into a 384 well plate. An equimolar amount of gRNA diluted insolutions with varied pH and salt is then added. After incubating atroom temperature for 10′ and brief centrifugation to remove any bubbles,a Bio-Rad CFX384™ Real-Time System C1000 Touch™ Thermal Cycler with theBio-Rad CFX Manager software is used to run a gradient from 20° C. to90° C. with a 10 increase in temperature every 10 seconds.

The second assay consists of mixing various concentrations of gRNA with2 uM Cas9 in optimal buffer from assay 1 above and incubating at RT for10′ in a 384 well plate. An equal volume of optimal buffer+10× SYPROOrange® (Life Technologies cat #S-6650) is added and the plate sealedwith Microseal® B adhesive (MSB-1001). Following brief centrifugation toremove any bubbles, a Bio-Rad CFX384™ Real-Time System C1000 Touch™Thermal Cycler with the Bio-Rad CFX Manager software is used to run agradient from 20° C. to 90° C. with a 10 increase in temperature every10 seconds.

VI. Genome Editing Approaches

While not wishing to be bound by theory, altering the LCA10 targetposition may be achieved using one of the approaches discussed herein.

NHEJ Approaches for Gene Targeting

As described herein, nuclease-induced non-homologous end-joining (NHEJ)can be used to introduce indels at a target position. Nuclease-inducedNHEJ can also be used to remove (e.g., delete) genomic sequenceincluding the mutation at a target position in a gene of interest.

While not wishing to be bound by theory, it is believed that, in anembodiment, the genomic alterations associated with the methodsdescribed herein rely on nuclease-induced NHEJ and the error-pronenature of the NHEJ repair pathway. NHEJ repairs a double-strand break inthe DNA by joining together the two ends; however, generally, theoriginal sequence is restored only if two compatible ends, exactly asthey were formed by the double-strand break, are perfectly ligated. TheDNA ends of the double-strand break are frequently the subject ofenzymatic processing, resulting in the addition or removal ofnucleotides, at one or both strands, prior to rejoining of the ends.This results in the presence of insertion and/or deletion (indel)mutations in the DNA sequence at the site of the NHEJ repair.

The indel mutations generated by NHEJ are unpredictable in nature;however, at a given break site certain indel sequences are favored andare over represented in the population, likely due to small regions ofmicrohomology. The lengths of deletions can vary widely; most commonlyin the 1-50 bp range, but they can easily reach greater than 100-200 bp.Insertions tend to be shorter and often include short duplications ofthe sequence immediately surrounding the break site. However, it ispossible to obtain large insertions, and in these cases, the insertedsequence has often been traced to other regions of the genome or toplasmid DNA present in the cells.

Because NHEJ is a mutagenic process, it can also be used to delete smallsequence motifs as long as the generation of a specific final sequenceis not required. If a double-strand break is targeted near to a shorttarget sequence, the deletion mutations caused by the NHEJ repair oftenspan, and therefore remove, the unwanted nucleotides. For the deletionof larger DNA segments, introducing two double-strand breaks, one oneach side of the sequence, can result in NHEJ between the ends withremoval of the entire intervening sequence. Both of these approaches canbe used to delete specific DNA sequences; however, the error-pronenature of NHEJ may still produce indel mutations at the site ofdeletion.

Both double strand cleaving eaCas9 molecules and single strand, ornickase, eaCas9 molecules can be used in the methods and compositionsdescribed herein to generate break-induced indels.

Double Strand Break

In an embodiment, double strand cleavage is effected by a Cas9 moleculehaving cleavage activity associated with an HNH-like domain and cleavageactivity associated with a RuvC-like domain, e.g., an N-terminalRuvC-like domain, e.g., a wild type Cas9. Such embodiments require onlya single gRNA.

Single Strand Break

In other embodiments, two single strand breaks are effected by a Cas9molecule having nickase activity, e.g., cleavage activity associatedwith an HNH-like domain or cleavage activity associated with anN-terminal RuvC-like domain. Such embodiments require two gRNAs, one forplacement of each single strand break. In an embodiment, the Cas9molecule having nickase activity cleaves the strand to which the gRNAhybridizes, but not the strand that is complementary to the strand towhich the gRNA hybridizes. In an embodiment, the Cas9 molecule havingnickase activity does not cleave the strand to which the gRNAhybridizes, but rather cleaves the strand that is complementary to thestrand to which the gRNA hybridizes.

In an embodiment, the nickase has HNH activity, e.g., a Cas9 moleculehaving the RuvC activity inactivated, e.g., a Cas9 molecule having amutation at D10, e.g., the D10A mutation. D10A inactivates RuvCtherefore the Cas9 nickase has (only) HNH activity and will cut on thestrand to which the gRNA hybridizes (the complementary strand, whichdoes not have the NGG PAM on it). In other embodiments, a Cas9 moleculehaving an H840, e.g., an H840A, mutation can be used as a nickase. H840Ainactivates HNH therefore the Cas9 nickase has (only) RuvC activity andcuts on the non-complementary strand (the strand that has the NGG PAMand whose sequence is identical to the gRNA). In other embodiments, aCas9 molecule having an H863, e.g., an H863A, mutation can be used as anickase. H863A inactivates HNH therefore the Cas9 nickase has (only)RuvC activity and cuts on the non-complementary strand (the strand thathas the NGG PAM and whose sequence is identical to the gRNA).

In an embodiment, in which a nickase and two gRNAs are used to positiontwo single strand breaks, one nick is on the + strand and one nick is onthe − strand of the target nucleic acid. The PAMs can be outwardlyfacing. The gRNAs can be selected such that the gRNAs are separated by,from 0-50, 0-100, or 0-200 nucleotides. In an embodiment, there is nooverlap between the target sequences that are complementary to thetargeting domains of the two gRNAs. In an embodiment, the gRNAs do notoverlap and are separated by as much as 50, 100, or 200 nucleotides. Inan embodiment, the use of two gRNAs can increase specificity, e.g., bydecreasing off-target binding (Ran 2013).

Placement of Double Strand or Single Strand Breaks Relative to theTarget Position

In an embodiment, in which a gRNA and Cas9 nuclease generate a doublestrand break for the purpose of inducing break-induced indels, a gRNA,e.g., a unimolecular (or chimeric) or modular gRNA molecule, isconfigured to position one double-strand break in close proximity to anucleotide of the target position. In an embodiment, the cleavage siteis between 0-40 bp away from the target position (e.g., less than 40,35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the targetposition).

In an embodiment, in which two gRNAs complexing with a Cas9 nickaseinduce two single strand breaks for the purpose of introducingbreak-induced indels, two gRNAs, e.g., independently, unimolecular (orchimeric) or modular gRNA, are configured to position two single-strandbreaks to provide for NHEJ-mediated alteration of a nucleotide of thetarget position. In an embodiment, the gRNAs are configured to positioncuts at the same position, or within a few nucleotides of one another,on different strands, essentially mimicking a double strand break. In anembodiment, the two nicks are between 0-40 bp away from the targetposition (e.g., less than 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4,3, 2 or 1 bp from the target position) respectively, and the two singlestrand breaks are within 25-55 bp of each other (e.g., between 25 to 50,25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 35 to 45, or 40to 45 bp) and no more than 100 bp away from each other (e.g., no morethan 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp). In an embodiment, thegRNAs are configured to place a single strand break on either side ofthe target position. In an embodiment, the gRNAs are configured to placea single strand break on the same side (either 5′ or 3′) of the targetposition.

Regardless of whether a break is a double strand or a single strandbreak, the gRNA should be configured to avoid unwanted target chromosomeelements, such as repeated elements, e.g., an Alu repeat, in the targetdomain. In addition, a break, whether a double strand or a single strandbreak, should be sufficiently distant from any sequence that should notbe altered. For example, cleavage sites positioned within introns shouldbe sufficiently distant from any intron/exon border, or naturallyoccurring splice signal, to avoid alteration of the exonic sequence orunwanted splicing events.

Single-Strand Annealing

Single strand annealing (SSA) is another DNA repair process that repairsa double-strand break between two repeat sequences present in a targetnucleic acid. Repeat sequences utilized by the SSA pathway are generallygreater than 30 nucleotides in length. Resection at the break endsoccurs to reveal repeat sequences on both strands of the target nucleicacid. After resection, single strand overhangs containing the repeatsequences are coated with RPA protein to prevent the repeats sequencesfrom inappropriate annealing, e.g., to themselves. RAD52 binds to andeach of the repeat sequences on the overhangs and aligns the sequencesto enable the annealing of the complementary repeat sequences. Afterannealing, the single-strand flaps of the overhangs are cleaved. New DNAsynthesis fills in any gaps, and ligation restores the DNA duplex. As aresult of the processing, the DNA sequence between the two repeats isdeleted. The length of the deletion can depend on many factors includingthe location of the two repeats utilized, and the pathway orprocessivity of the resection.

In contrast to HDR pathways, SSA does not require a template nucleicacid to alter or correct a target nucleic acid sequence. Instead, thecomplementary repeat sequence is utilized.

Other DNA Repair Pathways

SSBR (Single Strand Break Repair)

Single-stranded breaks (SSB) in the genome are repaired by the SSBRpathway, which is a distinct mechanism from the DSB repair mechanismsdiscussed above. The SSBR pathway has four major stages: SSB detection,DNA end processing, DNA gap filling, and DNA ligation. A more detailedexplanation is given in Caldecott, Nature Reviews Genetics 9, 619-631(August 2008), and a summary is given here.

In the first stage, when a SSB forms, PARP1 and/or PARP2 recognize thebreak and recruit repair machinery. The binding and activity of PARP1 atDNA breaks is transient and it seems to accelerate SSBr by promoting thefocal accumulation or stability of SSBr protein complexes at the lesion.Arguably the most important of these SSBr proteins is XRCC1, whichfunctions as a molecular scaffold that interacts with, stabilizes, andstimulates multiple enzymatic components of the SSBr process includingthe protein responsible for cleaning the DNA 3′ and 5′ ends. Forinstance, XRCC1 interacts with several proteins (DNA polymerase beta,PNK, and three nucleases, APE1, APTX, and APLF) that promote endprocessing. APE1 has endonuclease activity. APLF exhibits endonucleaseand 3′ to 5′ exonuclease activities. APTX has endonuclease and 3′ to 5′exonuclease activity.

This end processing is an important stage of SSBR since the 3′- and/or5′-termini of most, if not all, SSBs are ‘damaged’. End processinggenerally involves restoring a damaged 3′-end to a hydroxylated stateand and/or a damaged 5′ end to a phosphate moiety, so that the endsbecome ligation-competent. Enzymes that can process damaged 3′ terminiinclude PNKP, APE1, and TDP1. Enzymes that can process damaged 5′termini include PNKP, DNA polymerase beta, and APTX. LIG3 (DNA ligaseIII) can also participate in end processing. Once the ends are cleaned,gap filling can occur.

At the DNA gap filling stage, the proteins typically present are PARP1,DNA polymerase beta, XRCC1, FEN1 (flap endonuclease 1), DNA polymerasedelta/epsilon, PCNA, and LIG1. There are two ways of gap filling, theshort patch repair and the long patch repair. Short patch repairinvolves the insertion of a single nucleotide that is missing. At someSSBs, “gap filling” might continue displacing two or more nucleotides(displacement of up to 12 bases have been reported). FEN1 is anendonuclease that removes the displaced 5′-residues. Multiple DNApolymerases, including Pol β, are involved in the repair of SSBs, withthe choice of DNA polymerase influenced by the source and type of SSB.

In the fourth stage, a DNA ligase such as LIG1 (Ligase I) or LIG3(Ligase III) catalyzes joining of the ends. Short patch repair usesLigase III and long patch repair uses Ligase I.

Sometimes, SSBR is replication-coupled. This pathway can involve one ormore of CtIP, MRN, ERCC1, and FEN1. Additional factors that may promoteSSBR include: aPARP, PARP1, PARP2, PARG, XRCC1, DNA polymerase b, DNApolymerase d, DNA polymerase e, PCNA, LIG1, PNK, PNKP, APE1, APTX, APLF,TDP1, LIG3, FEN1, CtIP, MRN, and ERCC1.

MMR (Mismatch Repair)

Cells contain three excision repair pathways: MMR, BER, and NER. Theexcision repair pathways have a common feature in that they typicallyrecognize a lesion on one strand of the DNA, then exo/endonucleasesremove the lesion and leave a 1-30 nucleotide gap that issub-sequentially filled in by DNA polymerase and finally sealed withligase. A more complete picture is given in Li 2008, and a summary isprovided here.

Mismatch repair (MMR) operates on mispaired DNA bases.

The MSH2/6 or MSH2/3 complexes both have ATPases activity that plays animportant role in mismatch recognition and the initiation of repair.MSH2/6 preferentially recognizes base-base mismatches and identifiesmispairs of 1 or 2 nucleotides, while MSH2/3 preferentially recognizeslarger ID mispairs.

hMLH1 heterodimerizes with hPMS2 to form hMutLa which possesses anATPase activity and is important for multiple steps of MMR. It possessesa PCNA/replication factor C (RFC)-dependent endonuclease activity whichplays an important role in 3′ nick-directed MMR involving EXO1 (EXO1 isa participant in both HR and MMR). It regulates termination ofmismatch-provoked excision. Ligase I is the relevant ligase for thispathway. Additional factors that may promote MMR include: EXO1, MSH2,MSH3, MSH6, MLH1, PMS2, MLH3, DNA Pol d, RPA, HMGB1, RFC, and DNA ligaseI.

Base Excision Repair (BER)

The base excision repair (BER) pathway is active throughout the cellcycle; it is responsible primarily for removing small,non-helix-distorting base lesions from the genome. In contrast, therelated Nucleotide Excision Repair pathway (discussed in the nextsection) repairs bulky helix-distorting lesions. A more detailedexplanation is given in Caldecott, Nature Reviews Genetics 9, 619-631(August 2008), and a summary is given here.

Upon DNA base damage, base excision repair (BER) is initiated and theprocess can be simplified into five major steps: (a) removal of thedamaged DNA base; (b) incision of the subsequent a basic site; (c)clean-up of the DNA ends; (d) insertion of the correct nucleotide intothe repair gap; and (e) ligation of the remaining nick in the DNAbackbone. These last steps are similar to the SSBR.

In the first step, a damage-specific DNA glycosylase excises the damagedbase through cleavage of the N-glycosidic bond linking the base to thesugar phosphate backbone. Then AP endonuclease-1 (APE1) or bifunctionalDNA glycosylases with an associated lyase activity incised thephosphodiester backbone to create a DNA single strand break (SSB). Thethird step of BER involves cleaning-up of the DNA ends. The fourth stepin BER is conducted by Pol β that adds a new complementary nucleotideinto the repair gap and in the final step XRCC1/Ligase III seals theremaining nick in the DNA backbone. This completes the short-patch BERpathway in which the majority (˜80%) of damaged DNA bases are repaired.However, if the 5′-ends in step 3 are resistant to end processingactivity, following one nucleotide insertion by Pol β there is then apolymerase switch to the replicative DNA polymerases, Pol δ/ε, whichthen add ˜2-8 more nucleotides into the DNA repair gap. This creates a5′-flap structure, which is recognized and excised by flapendonuclease-1 (FEN-1) in association with the processivity factorproliferating cell nuclear antigen (PCNA). DNA ligase I then seals theremaining nick in the DNA backbone and completes long-patch BER.Additional factors that may promote the BER pathway include: DNAglycosylase, APE1, Polb, Pold, Pole, XRCC1, Ligase III, FEN-1, PCNA,RECQL4, WRN, MYH, PNKP, and APTX.

Nucleotide Excision Repair (NER)

Nucleotide excision repair (NER) is an important excision mechanism thatremoves bulky helix-distorting lesions from DNA. Additional detailsabout NER are given in Marteijn 2014, and a summary is given here. NER abroad pathway encompassing two smaller pathways: global genomic NER(GG-NER) and transcription coupled repair NER (TC-NER). GG-NER andTC-NER use different factors for recognizing DNA damage. However, theyutilize the same machinery for lesion incision, repair, and ligation.

Once damage is recognized, the cell removes a short single-stranded DNAsegment that contains the lesion. Endonucleases XPF/ERCC1 and XPG(encoded by ERCC5) remove the lesion by cutting the damaged strand oneither side of the lesion, resulting in a single-strand gap of 22-30nucleotides. Next, the cell performs DNA gap filling synthesis andligation. Involved in this process are: PCNA, RFC, DNA Pol δ, DNA Pol εor DNA Pol κ, and DNA ligase I or XRCC1/Ligase III. Replicating cellstend to use DNA pol ε and DNA ligase I, while non-replicating cells tendto use DNA Pol δ, DNA Pol κ, and the XRCC1/Ligase III complex to performthe ligation step.

NER can involve the following factors: XPA-G, POLH, XPF, ERCC1, XPA-G,and LIG1. Transcription-coupled NER (TC-NER) can involve the followingfactors: CSA, CSB, XPB, XPD, XPG, ERCC1, and TTDA. Additional factorsthat may promote the NER repair pathway include XPA-G, POLH, XPF, ERCC1,XPA-G, LIG1, CSA, CSB, XPA, XPB, XPC, XPD, XPF, XPG, TTDA, UVSSA, USP7,CETN2, RAD23B, UV-DDB, CAK subcomplex, RPA, and PCNA.

Interstrand Crosslink (ICL)

A dedicated pathway called the ICL repair pathway repairs interstrandcrosslinks. Interstrand crosslinks, or covalent crosslinks between basesin different DNA strand, can occur during replication or transcription.ICL repair involves the coordination of multiple repair processes, inparticular, nucleolytic activity, translesion synthesis (TLS), and HDR.Nucleases are recruited to excise the ICL on either side of thecrosslinked bases, while TLS and HDR are coordinated to repair the cutstrands. ICL repair can involve the following factors: endonucleases,e.g., XPF and RAD51C, endonucleases such as RAD51, translesionpolymerases, e.g., DNA polymerase zeta and Rev1), and the Fanconi anemia(FA) proteins, e.g., FancJ.

Other Pathways

Several other DNA repair pathways exist in mammals.

Translesion synthesis (TLS) is a pathway for repairing a single strandedbreak left after a defective replication event and involves translesionpolymerases, e.g., DNA pol □ and Rev1.

Error-free postreplication repair (PRR) is another pathway for repairinga single stranded break left after a defective replication event.

Examples of gRNAs in Genome Editing Methods

gRNA molecules as described herein can be used with Cas9 molecules thatcleave both or a single strand to alter the sequence of a target nucleicacid, e.g., of a target position or target genetic signature. gRNAmolecules useful in these method are described below.

In an embodiment, the gRNA, e.g., a chimeric gRNA, molecule isconfigured such that it comprises one or more of the followingproperties;

a) it can position, e.g., when targeting a Cas9 molecule that makesdouble strand breaks, a double strand break (i) within 50, 100, 150 or200 nucleotides of a target position, or (ii) sufficiently close thatthe target position is within the region of end resection;

b) it has a targeting domain of at least 17 nucleotides, e.g., atargeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides; and

c)

-   -   (i) the proximal and tail domain, when taken together, comprise        at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53        nucleotides, e.g., at least 15, 18, 20, 25, 30, 31, 35, 40, 45,        49, 50, or 53 nucleotides from a naturally occurring S.        pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail        and proximal domain, or a sequence that differs by no more than        1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;    -   (ii) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49,        50, or 53 nucleotides 3′ to the last nucleotide of the second        complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31,        35, 40, 45, 49, 50, or 53 nucleotides from the corresponding        sequence of a naturally occurring S. pyogenes, S.        thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence        that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10        nucleotides therefrom;    -   (iii) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50,        51, or 54 nucleotides 3′ to the last nucleotide of the second        complementarity domain that is complementary to its        corresponding nucleotide of the first complementarity domain,        e.g., at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54        nucleotides from the corresponding sequence of a naturally        occurring S. pyogenes, S. thermophilus, S. aureus, or N.        meningitidis gRNA, or a sequence that differs by no more than 1,        2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;    -   iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40        nucleotides in length, e.g., it comprises at least 10, 15, 20,        25, 30, 35 or 40 nucleotides from a naturally occurring S.        pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail        domain; or, or a sequence that differs by no more than 1, 2, 3,        4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or    -   (v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides        or all of the corresponding portions of a naturally occurring        tail domain, e.g., a naturally occurring S. pyogenes, S.        thermophilus, S. aureus, or N. meningitidis tail domain.

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: a(i); and b(i).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: a(i); and b(ii).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: a(i); and b(iii).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: a(ii); and b(i).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: a(ii); and b(ii).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: a(ii); and b(iii).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: b(i); and c(i).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: b(i); and c(ii).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: b(ii); and c(i).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: b(ii); and c(ii).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: b(iii); and c(i).

In an embodiment, the gRNA molecule is configured such that it comprisesproperties: b(iii); and c(ii).

In an embodiment, the gRNA is used with a Cas9 nickase molecule havingHNH activity, e.g., a Cas9 molecule having the RuvC activityinactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., theD10A mutation.

In an embodiment, the gRNA is used with a Cas9 nickase molecule havingRuvC activity, e.g., a Cas9 molecule having the HNH activityinactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., aH840A.

In an embodiment, the gRNA is used with a Cas9 nickase molecule havingRuvC activity, e.g., a Cas9 molecule having the HNH activityinactivated, e.g., a Cas9 molecule having a mutation at H863, e.g., aH863A.

In an embodiment, a pair of gRNA molecules, e.g., a pair of chimericgRNA molecules, comprising a first and a second gRNA molecule, isconfigured such that they comprises one or more of the followingproperties:

a) the first and second gRNA molecules position, e.g., when targeting aCas9 molecule that makes single strand or double strand breaks:

-   -   (i) as positioned by a first and second gRNA molecule described        herein; or    -   (ii) sufficiently close that the target position is altered when        the break is repaired;

b) one or both, independently, has a targeting domain of at least 17nucleotides, e.g., a targeting domain of (i) 17, (ii) 18, or (iii) 20nucleotides; and

c) one or both, independently, has a the tail domain is (i) at least 10,15, 20, 25, 30, 35 or 40 nucleotides in length or (ii) the tail domaincomprises, 15, 20, 25, 30, 35, 40, or all of the corresponding portionsof a naturally occurring tail domain, e.g., a naturally occurring S.pyogenes, S. aureus, or S. thermophilus tail domain.

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: a(i); and b(i).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: a(i); and b(ii).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: a(i); and b(iii).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: a(ii); and b(i).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: a(ii); and b(ii).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: a(ii); and b(iii).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: b(i); and c(i).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: b(i); and c(ii).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: b(ii); and c(i).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: b(ii); and c(ii).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: b(iii); and c(i).

In an embodiment, one or both of the gRNA molecules is configured suchthat it comprises properties: b(iii); and c(ii).

In an embodiment the gRNA is used with a Cas9 nickase molecule havingHNH activity, e.g., a Cas9 molecule having the RuvC activityinactivated, e.g., a Cas9 molecule having a mutation at D10, e.g., theD10A mutation.

In an embodiment, the gRNA is used with a Cas9 nickase molecule havingRuvC activity, e.g., a Cas9 molecule having the HNH activityinactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., aH840A.

In an embodiment the gRNA is used with a Cas9 nickase molecule havingRuvC activity, e.g., a Cas9 molecule having the HNH activityinactivated, e.g., a Cas9 molecule having a mutation at H863, e.g., aH863A.

Targets: Cells

Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA moleculecomplex, can be used to manipulate a cell, e.g., to edit a targetnucleic acid, in a wide variety of cells.

In some embodiments, a cell is manipulated by altering one or moretarget genes, e.g., as described herein. In some embodiments, theexpression of one or more target genes (e.g., one or more target genesdescribed herein) is modulated, e.g., in vivo.

In an embodiment, the target cell is a retinal cell, e.g., a cell of theretinal pigment epithelium cell or a photoreceptor cell. In anotherembodiment, the target cell is a horizontal cell, a bipolar cell, anamacrine cell, or a ganglion cell. In an embodiment, the target cell isa cone photoreceptor cell or cone cell, a rod photoreceptor cell or rodcell, or a macular cone photoreceptor cell. In an exemplary embodiment,cone photoreceptors in the macula are targeted, i.e., conephotoreceptors in the macula are the target cells.

In an embodiment, the target cell is removed from the subject, the genealtered ex vivo, and the cell returned to the subject. In an embodiment,a photoreceptor cell is removed from the subject, the gene altered exvivo, and the photoreceptor cell returned to the subject. In anembodiment, a cone photoreceptor cell is removed from the subject, thegene altered ex vivo, and the cone photoreceptor cell returned to thesubject.

In an embodiment, the cells are induced pluripotent stem cells (iPS)cells or cells derived from iPS cells, e.g., iPS cells from the subject,modified to alter the gene and differentiated into retinal progenitorcells or retinal cells, e.g., retinal photoreceptors, and injected intothe eye of the subject, e.g., subretinally, e.g., in the submacularregion of the retina.

In an embodiment, the cells are targeted in vivo, e.g., by delivery ofthe components, e.g., a Cas9 molecule and a gRNA molecule, to the targetcells. In an embodiment, the target cells are retinal pigmentepithelium, photoreceptor cells, or a combination thereof. In anembodiment, AAV is used to deliver the components, e.g., a Cas9 moleculeand a gRNA molecule, e.g., by transducing the target cells.

VII. Delivery, Formulations and Routes of Administration

The components, e.g., a Cas9 molecule and gRNA molecule can bedelivered, formulated, or administered in a variety of forms, see, e.g.,Table 18. In an embodiment, one Cas9 molecule and two or more (e.g., 2,3, 4, or more) different gRNA molecules are delivered, e.g., by an AAVvector. In an embodiment, the sequence encoding the Cas9 molecule andthe sequence(s) encoding the two or more (e.g., 2, 3, 4, or more)different gRNA molecules are present on the same nucleic acid molecule,e.g., an AAV vector. When a Cas9 or gRNA component is delivered encodedin DNA the DNA will typically include a control region, e.g., comprisinga promoter, to effect expression. Useful promoters for Cas9 moleculesequences include CMV, EFS, EF-1a, MSCV, PGK, CAG, hGRK1, hCRX, hNRL,and hRCVRN control promoters. In an embodiment, the promoter is aconstitutive promoter. In another embodiment, the promoter is a tissuespecific promoter. Exemplary promoter sequences are disclosed in Table20. Useful promoters for gRNAs include H1, 7SK, and U6 promoters.Promoters with similar or dissimilar strengths can be selected to tunethe expression of components. Sequences encoding a Cas9 molecule cancomprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In anembodiment, the sequence encoding a Cas9 molecule comprises at least twonuclear localization signals. In an embodiment a promoter for a Cas9molecule or a gRNA molecule can be, independently, inducible, tissuespecific, or cell specific. To detect the expression of a Cas9, anaffinity tag can be used. Useful affinity tag sequences include, but arenot limited to, 3×Flag tag, single Flag tag, HA tag, Myc tag or HIS tag.Exemplary affinity tag sequences are disclosed in Table 26. To regulateCas9 expression, e.g., in mammalian cells, polyadenylation signals(poly(A) signals) can be used. Exemplary polyadenylation signals aredisclosed in Table 27.

Table 18 provides examples of how the components can be formulated,delivered, or administered.

TABLE 18 Elements Cas9 gRNA Molecule(s) molecule(s) Comments DNA DNA Inthis embodiment a Cas9 molecule, typically an eaCas9 molecule, and agRNA are transcribed from DNA. In this embodiment they are encoded onseparate molecules. DNA In this embodiment a Cas9 molecule, typically aneaCas9 molecule, and a gRNA are transcribed from DNA, here from a singlemolecule. DNA RNA In this embodiment a Cas9 molecule, typically aneaCas9 molecule, is transcribed from DNA. mRNA RNA In this embodiment aCas9 molecule, typically an eaCas9 molecule, is transcribed from DNA.Protein DNA In this embodiment a Cas9 molecule, typically an eaCas9molecule, is provided as a protein. A gRNA is transcribed from DNA.Protein RNA In this embodiment an eaCas9 molecule is provided as aprotein.

Table 19 summarizes various delivery methods for the components of a Cassystem, e.g., the Cas9 molecule component and the gRNA moleculecomponent, as described herein.

TABLE 19 Delivery into Non- Duration Type of Dividing of Ex- GenomeMolecule Delivery Vector/Mode Cells pression Integration DeliveredPhysical (e.g., YES Transient NO Nucleic electroporation, Acids andparticle gun, Calcium Proteins Phosphate transfection) Viral RetrovirusNO Stable YES RNA Lentivirus YES Stable YES/NO RNA with modi- ficationsAdenovirus YES Transient NO DNA Adeno- YES Stable NO DNA AssociatedVirus (AAV) Vaccinia YES Very NO DNA Virus Transient Herpes YES StableNO DNA Simplex Virus Non- Cationic YES Transient Depends Nucleic ViralLiposomes on what is Acids and delivered Proteins Polymeric YESTransient Depends Nucleic Nanoparticles on what is Acids and deliveredProteins Biological Attenuated YES Transient NO Nucleic Non- BacteriaAcids Viral Engineered YES Transient NO Nucleic Delivery BacteriophagesAcids Vehicles Mammalian YES Transient NO Nucleic Virus-like AcidsParticles Biological YES Transient NO Nucleic liposomes: AcidsErythrocyte Ghosts and Exosomes

Table 20 describes exemplary promoter sequences that can be used in AAVvectors, e.g., for Cas9 expression.

TABLE 20 Cas9 Promoter Sequences Promoter Length (bp) DNA Sequence CMV617 SEQ ID NO: 401 EFS 252 SEQ ID NO: 402 Human GRK1 292 SEQ ID NO: 403(rhodopsin kinase) Human CRX (cone 113 SEQ ID NO: 404 rod homeoboxtranscription factor) Human NRL (neural 281 SEQ ID NO: 405 retinaleucine zipper transcription factor enhance upstream of the human TKterminal promoter) Human RCVRN 235 SEQ ID NO: 406 (recoverin)

Table 26 describes exemplary affinity tag sequences that can be used inAAV vectors, e.g., for Cas9 expression.

TABLE 26 Affinity tag Amino Acid Sequence 3XFlag tagDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 426) Flag tag (single) DYKDDDDK (SEQID NO: 451) HA tag YPYDVPDYA (SEQ ID NO: 452) Myc tag EQKLISEEDL (SEQ IDNO: 453) HIS tag HHHHHH (SEQ ID NO: 454)

Table 27 describes exemplary polyadenylation (polyA) sequences that canbe used in AAV vectors, e.g., for Cas9 expression.

TABLE 27 Exemplary PolyA Sequences PolyA DNA sequence Mini polyA SEQ IDNO: 424 bGH polyA SEQ ID NO: 455 SV40 polyA SEQ ID NO: 456

Table 25 describes exemplary Inverted Terminal Repeat (ITR) sequencesthat can be used in AAV vectors.

TABLE 25 Sequences of ITRs from Exemplary AAV Serotypes AAV SerotypeLeft ITR Sequence Right ITR Sequence AAV1 SEQ ID NO: 407 SEQ ID NO: 436AAV2 SEQ ID NO: 408 SEQ ID NO: 437 AAV3B SEQ ID NO: 409 SEQ ID NO: 438AAV4 SEQ ID NO: 410 SEQ ID NO: 439 AAV5 SEQ ID NO: 411 SEQ ID NO: 440AAV6 SEQ ID NO: 412 SEQ ID NO: 441 AAV7 SEQ ID NO: 413 SEQ ID NO: 442AAV8 SEQ ID NO: 414 SEQ ID NO: 443 AAV9 SEQ ID NO: 415 SEQ ID NO: 444

Additional exemplary sequences for the recombinant AAV genome componentsdescribed herein are provided below.

Exemplary left and right ITR sequences are provided in Table 25 (SEQ IDNOs: 407-415 and 436-444).

Exemplary spacer 1 sequence: (SEQ ID NO: 416) CAGATCTGAATTCGGTACC.Exemplary U6 promoter sequence: (SEQ ID NO: 417)AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGA AAGGACGAAACACC

Exemplary gRNA targeting domain sequences are described herein, e.g., inTables 2A-2D, Tables 3A-3C, Tables 4A-4D, Tables 5A-5D, Tables 6A-6B,Tables 7A-7D, Tables 8A-8D, Tables 9A-9E, Tables 10A-10B, or Table 11.

Exemplary gRNA scaffold domain sequence: (SEQ ID NO: 418)GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTT. Exemplary spacer 2 domain sequence:(SEQ ID NO: 419) GGTACCGCTAGCGCTTAAGTCGCGATGTACGGGCCAGATATACGCGT TGA.Exemplary Polymerase II promoter sequences are provided in Table 20.Exemplary N-ter NLS nucleotide sequence: (SEQ ID NO: 420)CCGAAGAAAAAGCGCAAGGTCGAAGCGTCC Exemplary N-ter NLS amino acid sequence:(SEQ ID NO: 434) PKKKRKV Exemplary S. aureus Cas9 nucleotide sequenceset forth in SEQ ID NO: 39. Exemplary S. aureus Cas9 amino acid sequenceset forth in SEQ ID NO: 26. Exemplary C-ter NLS sequence: (SEQ ID NO:422) CCCAAGAAGAAGAGGAAAGTC. Exemplary C-ter NLS amino acid sequence:(SEQ ID NO: 434) PKKKRKV Exemplary poly(A) signal sequence: (SEQ ID NO:424) TAGCAATAAAGGATCGTTTATTTTCATTGGAAGCGTGTGTTGGTTTTTTG ATCAGGCGCG.Exemplary Spacer 3 sequence: (SEQ ID NO: 425)TCCAAGCTTCGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCGTTAACTCTAGATTTAAATGCATGCTGGGGAGAGATCT Exemplary 3xFLAG nucleotidesequence: (SEQ ID NO: 423)GACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAA GGATGACGATGACAAG.Exemplary 3xFLAG amino acid sequence: (SEQ ID NO: 426)DYKDHDGDYKDHDIDYKDDDDK Exemplary Spacer 4 sequence: (SEQ ID NO: 427)CGACTTAGTTCGATCGAAGG.

Exemplary recombinant AAV genome sequences are provided in FIGS. 19A-24F(SEQ ID NOs: 428-433 and 445-450). Exemplary sequences of therecombinant AAV genome components (e.g., one or more of the componentsdescribed above) are also shown in FIGS. 19A-24F (SEQ ID NOs: 428-433and 445-450).

In certain aspects, the present disclosure focuses on AAV vectorsencoding CRISPR/Cas9 genome editing systems, and on the use of suchvectors to treat CEP290 associated disease. Exemplary AAV vector genomesare schematized in FIGS. 25A through 25D, which illustrate certain fixedand variable elements of these vectors: inverted terminal repeats(ITRs), one or two gRNA sequences and promoter sequences to drive theirexpression, a Cas9 coding sequence and another promoter to drive itsexpression. Each of these elements is discussed in detail below.

Turning first to the gRNA pairs utilized in the nucleic acids or AAVvectors of the present disclosure, one of three “left” or “upstream”guides may be used to cut upstream (between exon 26 and the IVS26mutation), and one of three “right” or “downstream” guides is used tocut downstream (between the IVS26 mutation and exon 27). Targetingdomain sequences of these guides (both DNA and RNA sequences) arepresented in Table 28, below:

TABLE 28 Upstream (left) and Downstream (right) gRNA Targeting DomainSequences SEQ SEQ ID ID NO: DNA NO: RNA Upstream (left) guides 389GTTCTGTCCTCAGTAAAAGG 530 GUUCUGUCCUCAGUAAAAGGUA TA 390GAATAGTTTGTTCTGGGTAC 468 GAAUAGUUUGUUCUGGGUAC 391 GAGAAAGGGATGGGCACTTA538 GAGAAAGGGAUGGGCACUUA Downstream (right) guides 388GTCAAAAGCTACCGGTTACC 558 GUCAAAAGCUACCGGUUACCUG TG 392GATGCAGAACTAGTGTAGAC 460 GAUGCAGAACUAGUGUAGAC 394 GAGTATCTCCTGTTTGGCA568 GAGUAUCUCCUGUUUGGCA

The left and right guides can be used in any combination, though certaincombinations may be more suitable for certain applications. Table 29sets forth several upstream+downstream guide pairs used in theembodiments of this disclosure. It should be noted, notwithstanding theuse of “left” and “right” as nomenclature for gRNAs, that any guide in apair, upstream or downstream, may be placed in either one of the gRNAcoding sequence positions illustrated in FIG. 25.

TABLE 29 Upstream (Left) + Downstream (Right) Guide Pairs by SEQ ID NO.Downstream 388 392 394 Upstream 389 389 + 388 389 + 392 389 + 394 390390 + 388 390 + 392 390 + 394 391 391 + 388 391 + 392 391 + 394 530530 + 558 530 + 460 530 + 568 468 468 + 558 468 + 460 468 + 568 538538 + 558 538 + 460 538 + 568

In some embodiments, the gRNAs used in the present disclosure arederived from S. aureus gRNAs and can be unimolecular or modular, asdescribed below. An exemplary unimolecular S. aureus gRNA is shown inFIG. 18B, and exemplary DNA and RNA sequences corresponding tounimolecular S. aureus gRNAs are shown below:

(SEQ ID NO: 2785) DNA: [N]₁₆₋₂₄GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTT and (SEQ ID NO: 2779) RNA: [N]₁₆₋₂₄GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUUU. (SEQ ID NO: 2787) DNA: [N]₁₆₋₂₄GTTATAGTACTCTGGAAACAGAATCTACTATAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTT and (SEQ ID NO: 2786) RNA: [N]₁₆₋₂₄GUUAUAGUACUCUGGAAACAGAAUCUACUAUAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGAUUUUUU.

It should be noted that, while the figure depicts a targeting domain of20 nucleotides, the targeting domain can have any suitable length. gRNAsused in the various embodiments of this disclosure preferably includetargeting domains of between 16 and 24 (inclusive) bases in length attheir 5′ ends, and optionally include a 3′ U6 termination sequence asillustrated.

The gRNA in FIG. 18B is depicted as unimolecular, but in some instancesmodular guides can be used. In the exemplary unimolecular gRNA sequencesabove, a 5′ portion corresponding to a crRNA (underlined) is connectedby a GAAA linker to a 3′ portion corresponding to a tracrRNA (doubleunderlined). Skilled artisans will appreciate that two-part modulargRNAs can be used that correspond to the underlined and doubleunderlined sections.

Either one of the gRNAs presented above can be used with any oftargeting sequences SEQ ID NOs: 389-391, 388, 392, or 394, and two gRNAsin a pair do not necessarily include the same backbone sequence.Additionally, skilled artisans will appreciate that the exemplary gRNAdesigns set forth herein can be modified in a variety of ways, which aredescribed below or are known in the art; the incorporation of suchmodifications is within the scope of this disclosure.

Expression of each of the gRNAs in the AAV vector is driven by a pair ofU6 promoters, such as a human U6 promoter. An exemplary U6 promotersequence, as set forth in Maeder, is SEQ ID NO: 417.

Turning next to Cas9, in some embodiments the Cas9 protein is S. aureusCas9. In further embodiments of this disclosure an S. aureus Cas9sequence is modified to include two nuclear localization sequences(NLSs) at the C- and N-termini of the Cas9 protein, and amini-polyadenylation signal (or Poly-A sequence). Exemplary S. aureusCas9 sequences are provided as SEQ ID NO: 39 (i.e., codon-optimized S.aureus Cas9 nucleotide sequence) and SEQ ID NO: 26 (i.e., S. aureus Cas9protein sequence). These sequences are exemplary in nature, and are notintended to be limiting. The skilled artisan will appreciate thatmodifications of these sequences may be possible or desirable in certainapplications; such modifications are described below, or are known inthe art, and are within the scope of this disclosure.

Skilled artisans will also appreciate that polyadenylation signals arewidely used and known in the art, and that any suitable polyadenylationsignal can be used in the embodiments of this disclosure. One exemplarypolyadenylation signal is set forth in SEQ ID NO: 424.

Cas9 expression is driven, in certain vectors of this disclosure, by oneof three promoters: cytomegalovirus (CMV) (i.e., SEQ ID NO: 401),elongation factor-1 (EFS) (i.e., SEQ ID NO: 402), or human g-proteinreceptor coupled kinase-1 (hGRK1) (i.e., SEQ ID NO: 403), which isspecifically expressed in retinal photoreceptor cells. Modifications ofthe sequences of the promoters may be possible or desirable in certainapplications, and such modifications are within the scope of thisdisclosure.

AAV genomes according to the present disclosure generally incorporateinverted terminal repeats (ITRs) derived from the AAV2 serotype.Exemplary left and right ITRs are SEQ ID NO: 408 (AAV2 Left ITR) and SEQID NO: 437 (AAV2 Right ITR), respectively. It should be noted, however,that numerous modified versions of the AAV2 ITRs are used in the field,and the ITR sequences shown below are exemplary and are not intended tobe limiting. Modifications of these sequences are known in the art, orwill be evident to skilled artisans, and are thus included in the scopeof this disclosure.

As FIG. 25 illustrates, the gRNA pairs and the Cas9 promoter arevariable and can be selected from the lists presented above. Forclarity, this disclosure encompasses nucleic acids and/or AAV vectorscomprising any combination of these elements, though certaincombinations may be preferred for certain applications. Accordingly, invarious embodiments of this disclosure, a nucleic acid or AAV vectorencodes a CMV promoter for the Cas9, and gRNAs comprising targetingdomains according to SEQ ID NOs: 389 and 388 (RNA sequences are SEQ IDNOs: 530 and 558, respectively); a CMV promoter and gRNAs comprisingtargeting domains according to SEQ ID NOs: 389 and 392 (RNA sequencesare SEQ ID NOs: 530 and 460, respectively); a CMV promoter and gRNAscomprising targeting domains according to SEQ ID NOs: 389 and 394 (RNAsequences are SEQ ID NOs: 530 and 568, respectively); a CMV promoter andgRNAs comprising targeting domains according to SEQ ID NOs: 390 and 388(RNA sequences are SEQ ID NOs: 468 and 558, respectively); a CMVpromoter and gRNAs comprising targeting domains according to SEQ ID NOs:390 and 392 (RNA sequences are SEQ ID NOs: 468 and 460, respectively); aCMV promoter and gRNAs comprising targeting domains according to SEQ IDNOs: 390 and 394 (RNA sequences are SEQ ID NOs: 468 and 568,respectively); a CMV promoter and gRNAs comprising targeting domainsaccording to SEQ ID NOs: 391 and 388 (RNA sequences are SEQ ID NOs: 538and 558, respectively); a CMV promoter and gRNAs comprising targetingdomains according to SEQ ID NOs: 391 and 392 (RNA sequences are SEQ IDNOs: 538 and 460, respectively); a CMV promoter and gRNAs comprisingtargeting domains according to SEQ ID NOs: 391 and 394 (RNA sequencesare SEQ ID NOs: 538 and 568, respectively); an EFS promoter and gRNAscomprising targeting domains according to SEQ ID NOs: 389 and 388 (RNAsequences are SEQ ID NOs: 530 and 558, respectively); an EFS promoterand gRNAs comprising targeting domains according to SEQ ID NOs: 389 and392 (RNA sequences are SEQ ID NOs: 530 and 460, respectively); an EFSpromoter and gRNAs comprising targeting domains according to SEQ ID NOs:389 and 394 (RNA sequences are SEQ ID NOs: 530 and 568, respectively);an EFS promoter and gRNAs comprising targeting domains according to SEQID NOs: 390 and 388 (RNA sequences are SEQ ID NOs: 468 and 558,respectively); an EFS promoter and gRNAs comprising targeting domainsaccording to SEQ ID NOs: 390 and 392 (RNA sequences are SEQ ID NOs: 468and 460, respectively); an EFS promoter and gRNAs comprising targetingdomains according to SEQ ID NOs: 390 and 394 (RNA sequences are SEQ IDNOs: 468 and 568, respectively); an EFS promoter and gRNAs comprisingtargeting domains according to SEQ ID NOs: 391 and 388 (RNA sequencesare SEQ ID NOs: 538 and 558, respectively); an EFS promoter and gRNAscomprising targeting domains according to SEQ ID NOs: 391 and 392 (RNAsequences are SEQ ID NOs: 538 and 460, respectively); an EFS promoterand gRNAs comprising targeting domains according to SEQ ID NOs: 391 and394 (RNA sequences are SEQ ID NOs: 538 and 568, respectively); an hGRK1promoter and gRNAs comprising targeting domains according to SEQ ID NOs:389 and 388 (RNA sequences are SEQ ID NOs: 530 and 558, respectively);an hGRK1 promoter and gRNAs comprising targeting domains according toSEQ ID NOs: 389 and 392 (RNA sequences are SEQ ID NOs: 530 and 460,respectively); an hGRK1 promoter and gRNAs comprising targeting domainsaccording to SEQ ID NOs: 389 and 394 (RNA sequences are SEQ ID NOs: 530and 568, respectively); an hGRK1 promoter and gRNAs comprising targetingdomains according to SEQ ID NOs: 390 and 388 (RNA sequences are SEQ IDNOs: 468 and 558, respectively); an hGRK1 promoter and gRNAs comprisingtargeting domains according to SEQ ID NOs: 390 and 392 (RNA sequencesare SEQ ID NOs: 468 and 460, respectively); an hGRK1 promoter and gRNAscomprising targeting domains according to SEQ ID NOs: 390 and 394 (RNAsequences are SEQ ID NOs: 468 and 568, respectively); an hGRK1 promoterand gRNAs comprising targeting domains according to SEQ ID NOs: 391 and388 (RNA sequences are SEQ ID NOs: 538 and 558, respectively); an hGRK1promoter and gRNAs comprising targeting domains according to SEQ ID NOs:391 and 392 (RNA sequences are SEQ ID NOs: 538 and 460, respectively);or an hGRK1 promoter and gRNAs comprising targeting domains according toSEQ ID NOs: 391 and 394 (RNA sequences are SEQ ID NOs: 538 and 568,respectively).

In various embodiments, the nucleic acid or AAV vector encodes thefollowing: left and right AAV2 ITR sequences, a first U6 promoter todrive expression of a first guide RNA having a sequence selected fromSEQ ID NOs: 2785 and 2787 and/or comprising a targeting domain sequenceaccording to one of SEQ ID NOs: 389-391, a second U6 promoter to driveexpression of a second guide RNA comprising a sequence selected from SEQID NOs: 2785 and 2787 and/or comprising a targeting domain sequenceaccording to one of SEQ ID NOs: 388, 392, and 394, and a CMV promoter todrive expression of an S. aureus Cas9 encoded by SEQ ID NO: 39; or leftand right AAV2 ITR sequences, a first U6 promoter to drive expression ofa first guide RNA having a sequence selected from SEQ ID NOs: 2785 or2787 and/or comprising a targeting domain sequence according to one ofSEQ ID NOs: 389-391, a second U6 promoter to drive expression of asecond guide RNA comprising a sequence selected from SEQ ID NOs: 2785and 2787 and/or comprising a targeting domain sequence according to oneof SEQ ID NOs: 388, 392, and 394, and an hGRK promoter to driveexpression of an S. aureus Cas9 encoded by SEQ ID NO: 39; or left andright AAV2 ITR sequences, a first U6 promoter to drive expression of afirst guide RNA having a sequence selected from SEQ ID NOs: 2785 and2787 and/or comprising a targeting domain sequence according to one ofSEQ ID NOs: 389-391, a second U6 promoter to drive expression of asecond guide RNA comprising a sequence selected from SEQ ID NOs: 2785 or2787 and/or comprising a targeting domain sequence according to one ofSEQ ID NOs: 388, 392, and 394, and an EFS promoter to drive expressionof an S. aureus Cas9 encoded by SEQ ID NO: 39.

As shown in FIG. 25D, the nucleic acid or AAV vector may also comprise aSimian virus 40 (SV40) splice donor/splice acceptor (SD/SA) sequenceelement. In certain embodiments, the SV40 SD/SA element may bepositioned between the GRK1 promoter and the Cas9 gene. In certainembodiments, a Kozak consensus sequence may precede the start codon ofCas9 to ensure robust Cas9 expression.

In some embodiments, the nucleic acid or AAV vector shares at least 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity with oneof the nucleic acids or AAV vectors recited above.

It should be noted that these sequences described above are exemplary,and can be modified in ways that do not disrupt the operating principlesof elements they encode. Such modifications, some of which are discussedbelow, are within the scope of this disclosure. Without limiting theforegoing, skilled artisans will appreciate that the DNA, RNA or proteinsequences of the elements of this disclosure may be varied in ways thatdo not interrupt their function, and that a variety of similar sequencesthat are substantially similar (e.g., greater than 90%, 95%, 96%, 97%,98% or 99% sequence similarity, or in the case of short sequences suchas gRNA targeting domains, sequences that differ by no more than 1, 2 or3 nucleotides) can be utilized in the various systems, methods and AAVvectors described herein. Such modified sequences are within the scopeof this disclosure.

The AAV genomes described above can be packaged into AAV capsids (forexample, AAV5 capsids), which capsids can be included in compositions(such as pharmaceutical compositions) and/or administered to subjects.An exemplary pharmaceutical composition comprising an AAV capsidaccording to this disclosure can include a pharmaceutically acceptablecarrier such as balanced saline solution (BSS) and one or moresurfactants (e.g., Tween20) and/or a thermosensitive orreverse-thermosensitive polymer (e.g., pluronic). Other pharmaceuticalformulation elements known in the art may also be suitable for use inthe compositions described here.

Compositions comprising AAV vectors according to this disclosure can beadministered to subjects by any suitable means, including withoutlimitation injection, for example, subretinal injection. Theconcentration of AAV vector within the composition is selected toensure, among other things, that a sufficient AAV dose is administeredto the retina of the subject, taking account of dead volume within theinjection apparatus and the relatively limited volume that can be safelyadministered to the retina. Suitable doses may include, for example,1×10¹¹ viral genomes (vg)/mL, 2×10¹¹ viral genomes (vg)/mL, 3×10¹¹ viralgenomes (vg)/mL, 4×10¹¹ viral genomes (vg)/mL, 5×10¹¹ viral genomes(vg)/mL, 6×10¹¹ viral genomes (vg)/mL, 7×10¹¹ viral genomes (vg)/mL,8×10¹¹ viral genomes (vg)/mL, 9×10¹¹ viral genomes (vg)/mL, 1×10¹²vg/mL, 2×10¹² viral genomes (vg)/mL, 3×10¹² viral genomes (vg)/mL,4×10¹² viral genomes (vg)/mL, 5×10¹² viral genomes (vg)/mL, 6×10¹² viralgenomes (vg)/mL, 7×10¹² viral genomes (vg)/mL, 8×10¹² viral genomes(vg)/mL, 9×10¹² viral genomes (vg)/mL, 1×10¹³ vg/mL, 2×10¹³ viralgenomes (vg)/mL, 3×10¹³ viral genomes (vg)/mL, 4×10¹³ viral genomes(vg)/mL, 5×10¹³ viral genomes (vg)/mL, 6×10¹³ viral genomes (vg)/mL,7×10¹³ viral genomes (vg)/mL, 8×10¹³ viral genomes (vg)/mL, or 9×10¹³viral genomes (vg)/mL. Any suitable volume of the composition may bedelivered to the subretinal space. In some instances, the volume isselected to form a bleb in the subretinal space, for example 1microliter, 10 microliters, 50 microliters, 100 microliters, 150microliters, 200 microliters, 250 microliters, 300 microliters, etc. Anyregion of the retina may be targeted, though the fovea (which extendsapproximately 1 degree out from the center of the eye) may be preferredin certain instances due to its role in central visual acuity and therelatively high concentration of cone photoreceptors there relative toperipheral regions of the retina. Alternatively or additionally,injections may be targeted to parafoveal regions (extending betweenapproximately 2 and 10 degrees off center), which are characterized bythe presence of all three types of retinal photoreceptor cells. Inaddition, injections into the parafoveal region may be made atcomparatively acute angles using needle paths that cross the midline ofthe retina. For instance, injection paths may extend from the nasalaspect of the sclera near the limbus through the vitreal chamber andinto the parafoveal retina on the temporal side, from the temporalaspect of the sclera to the parafoveal retina on the nasal side, from aportion of the sclera located superior to the cornea to an inferiorparafoveal position, and/or from an inferior portion of the sclera to asuperior parafoveal position. The use of relatively small angles ofinjection relative to the retinal surface may advantageously reduce orlimit the potential for spillover of vector from the bleb into thevitreous body and, consequently, reduce the loss of the vector duringdelivery. In other cases, the macula (inclusive of the fovea) can betargeted, and in other cases, additional retinal regions can betargeted, or can receive spillover doses.

To mitigate ocular inflammation and associated discomfort, one or morecorticosteroids may be administered before, during, and/or afteradministration of the composition comprising AAV vectors. In certainembodiments, the corticosteroid may be an oral corticosteroid. Incertain embodiments, the oral corticosteroid may be prednisone. Incertain embodiments, the corticosteroid may be administered as aprophylactic, prior to administration of the composition comprising AAVvectors. For example, the corticosteroid may be administered the dayprior to administration, or 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 daysprior to administration of the composition comprising AAV vectors. Incertain embodiments, the corticosteroid may be administered for 1 weekto 10 weeks after administration of the composition comprising AAVvectors (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7weeks, 8 weeks, 9 weeks, or 10 weeks after administration of thecomposition comprising AAV vectors). In certain embodiments, thecorticosteroid treatment may be administered prior to (e.g., the dayprior to administration, or 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 daysprior to administration) and after administration of the compositioncomprising AAV vectors (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks afteradministration). For example, the corticosteroid treatment may beadministered beginning 3 days prior to until 6 weeks afteradministration of the AAV vector.

Suitable doses of corticosteroids may include, for example, 0.1mg/kg/day to 10 mg/kd/day (e.g., 0.1 mg/kg/day, 0.2 mg/kg/day, 0.3mg/kg/day, 0.4 mg/kg/day, 0.5 mg/kg/day, 0.6 mg/kg/day, 0.7 mg/kg/day,0.8 mg/kg/day, 0.9 mg/kg/day, or 1.0 mg/kg/day). In certain embodiments,the corticosteroid may be administered at an elevated dose during thecorticosteroid treatment, followed by a tapered dose of thecorticosteroid. For example, 0.5 mg/kg/day corticosteroid may beadministered for 4 weeks, followed by a 15-day taper (0.4 mg/kg/day for5 days, and then 0.2 mg/kg/day for 5 days, and then 0.1 mg/kg/day for 5days). The corticosteroid dose may be increased if there is an increasein vitreous inflammation by 1+ on the grading scale following surgery(e.g., within 4 weeks after surgery). For example, if there is anincrease in vitreous inflammation by 1+ on the grading scale while thepatient is receiving a 0.5 mg/kg/day dose (e.g., within 4 weeks aftersurgery), the corticosteroid dose may be may be increased to 1mg/kg/day. If any inflammation is present within 4 weeks after surgery,the taper may be delayed.

For pre-clinical development purposes, systems, compositions,nucleotides and vectors according to this disclosure can be evaluated exvivo using a retinal explant system, or in vivo using an animal modelsuch as a mouse, rabbit, pig, nonhuman primate, etc. Retinal explantsare optionally maintained on a support matrix, and AAV vectors can bedelivered by injection into the space between the photoreceptor layerand the support matrix, to mimic subretinal injection. Tissue forretinal explantation can be obtained from human or animal subjects, forexample mouse.

Explants are particularly useful for studying the expression of gRNAsand/or Cas9 following viral transduction, and for studying genomeediting over comparatively short intervals. These models also permithigher throughput than may be possible in animal models, and can bepredictive of expression and genome editing in animal models andsubjects. Small (mouse, rat) and large animal models (such as rabbit,pig, nonhuman primate) can be used for pharmacological and/ortoxicological studies and for testing the systems, nucleotides, vectorsand compositions of this disclosure under conditions and at volumes thatapproximate those that will be used in clinic. Because model systems areselected to recapitulate relevant aspects of human anatomy and/orphysiology, the data obtained in these systems will generally (thoughnot necessarily) be predictive of the behavior of AAV vectors andcompositions according to this disclosure in human and animal subjects.

While the foregoing exemplary embodiments have focused on guide RNAs,nucleic acids and AAV vectors targeted to the CEP290 gene, it will beappreciated by those of skill in the art that the nucleic acids andvectors of this disclosure may be used in the editing of other genetargets and the treatment of other diseases such as hereditaryretinopathies that may be treated by editing of genes other than CEP290.In certain embodiments, it is contemplated that the same configurationof the nucleic acids or AAV vectors disclosed herein may be used totreat other inherited retinal diseases by modifying the gRNA targetingdomains to target and alter the gene of interest.

FIGS. 25A-25D illustrate exemplary AAV vectors that may be used totransduce retinal cells, including without limitation retinalphotoreceptor cells such as rod photoreceptors and/or conephotoreceptors, and/or other retinal cell types. The AAV genome of FIG.25A comprises two guide RNAs comprising targeting domains according toone of the guide pairs of SEQ ID NOs: 389 and 388, SEQ ID NOs: 389 and392, SEQ ID NOs: 389 and 394, SEQ ID NOs: 390 and 388, SEQ ID NOs: 390and 392, SEQ ID NOs: 390 and 394, SEQ ID NOs: 391 and 388, SEQ ID NOs:391 and 392, SEQ ID NOs: 391 and 394, and a promoter sequence accordingto one of CMV, EFS, hGRK1 driving expression of an S. aureus Cas9comprising one or two nuclear localization signals and a polyadenylationsignal. The vector may additionally include ITRs such as AAV2 ITRs, orother sequences that may be selected for the specific application towhich the vector will be employed. A more detailed version of FIG. 25Ais shown in FIG. 25D, illustrating that the AAV genome may also comprisea Simian virus 40 (SV40) splice donor/splice acceptor (SD/SA) sequenceelement. In certain embodiments, the SV40 SD/SA element may bepositioned between the promoter and the Cas9 gene. In certainembodiments, a Kozak consensus sequence may precede the start codon ofCas9. The AAV genome of FIG. 25B comprises two guide RNAs according toSEQ ID NOs: 2785 or 2787, and a promoter sequence according to one ofSEQ ID NOs: 401-403 driving expression of an S. aureus Cas9 comprisingone or two nuclear localization signals and, optionally, apolyadenylation signal. The vector may additionally include ITRs such asAAV2 ITRs, or other sequences that may be selected for the specificapplication to which the vector will be employed. As is shown in FIG.25C, other vectors within the scope of this disclosure may include only1 guide RNA. Thus, in specific embodiments, an AAV genome of thisdisclosure may encode a CMV promoter for the Cas9 and one guide RNAhaving a sequence comprising, or sharing at least 90% sequence identitywith, a sequence selected from SEQ ID NOs: 2785 and 2787; a CMV promoterfor the Cas9 and two guide RNAs, each having a sequence comprising, orsharing at least 90% sequence identity with, a sequence selected fromSEQ ID NOs: 2785 and 2787; an hGRK promoter for the Cas9 and one guideRNA having a sequence comprising, or sharing at least 90% sequenceidentity with, a sequence selected from SEQ ID NOs: 2785 and 2787; anhGRK promoter for the Cas9 and two guide RNAs, each having a sequencecomprising, or sharing at least 90% sequence identity with, a sequenceselected from SEQ ID NOs: 2785 and 2787; an EFS promoter for the Cas9and one guide RNA having a sequence comprising, or sharing at least 90%sequence identity with, a sequence selected from SEQ ID NOs: 2785 and2787; an EFS promoter for the Cas9 and two guide RNAs, each having asequence comprising, or sharing at least 90% sequence identity with, asequence selected from SEQ ID NOs: 2785 and 2787.

DNA-Based Delivery of a Cas9 Molecule and/or a gRNA Molecule

Nucleic acids encoding Cas9 molecules (e.g., eaCas9 molecules) and/orgRNA molecules, can be administered to subjects or delivered into cellsby art-known methods or as described herein. For example, Cas9-encodingand/or gRNA-encoding DNA can be delivered, e.g., by vectors (e.g., viralor non-viral vectors), non-vector based methods (e.g., using naked DNAor DNA complexes), or a combination thereof.

DNA encoding Cas9 molecules (e.g., eaCas9 molecules) and/or gRNAmolecules can be conjugated to molecules (e.g., N-acetylgalactosamine)promoting uptake by the target cells (e.g., the target cells describedherein). Donor template molecules can be conjugated to molecules (e.g.,N-acetylgalactosamine) promoting uptake by the target cells (e.g., thetarget cells described herein).

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya vector (e.g., viral vector/virus or plasmid).

A vector can comprise a sequence that encodes a Cas9 molecule and/or agRNA molecule. A vector can also comprise a sequence encoding a signalpeptide (e.g., for nuclear localization, nucleolar localization,mitochondrial localization), fused, e.g., to a Cas9 molecule sequence.For example, a vector can comprise a nuclear localization sequence(e.g., from SV40) fused to the sequence encoding the Cas9 molecule.

One or more regulatory/control elements, e.g., a promoter, an enhancer,an intron, a polyadenylation signal, a Kozak consensus sequence,internal ribosome entry sites (IRES), a 2A sequence, and splice acceptoror donor can be included in the vectors. In some embodiments, thepromoter is recognized by RNA polymerase II (e.g., a CMV promoter). Inother embodiments, the promoter is recognized by RNA polymerase III(e.g., a U6 promoter). In some embodiments, the promoter is a regulatedpromoter (e.g., inducible promoter). In other embodiments, the promoteris a constitutive promoter. In some embodiments, the promoter is atissue specific promoter. In some embodiments, the promoter is a viralpromoter. In other embodiments, the promoter is a non-viral promoter.

In some embodiments, the vector or delivery vehicle is a viral vector(e.g., for generation of recombinant viruses). In some embodiments, thevirus is a DNA virus (e.g., dsDNA or ssDNA virus). In other embodiments,the virus is an RNA virus (e.g., an ssRNA virus). Exemplary viralvectors/viruses include, e.g., retroviruses, lentiviruses, adenovirus,adeno-associated virus (AAV), vaccinia viruses, poxviruses, and herpessimplex viruses.

In some embodiments, the virus infects dividing cells. In otherembodiments, the virus infects non-dividing cells. In some embodiments,the virus infects both dividing and non-dividing cells. In someembodiments, the virus can integrate into the host genome. In someembodiments, the virus is engineered to have reduced immunity, e.g., inhuman. In some embodiments, the virus is replication-competent. In otherembodiments, the virus is replication-defective, e.g., having one ormore coding regions for the genes necessary for additional rounds ofvirion replication and/or packaging replaced with other genes ordeleted. In some embodiments, the virus causes transient expression ofthe Cas9 molecule and/or the gRNA molecule. In other embodiments, thevirus causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanentexpression, of the Cas9 molecule and/or the gRNA molecule. The packagingcapacity of the viruses may vary, e.g., from at least about 4 kb to atleast about 30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.

In an embodiment, the viral vector recognizes a specific cell type ortissue. For example, the viral vector can be pseudotyped with adifferent/alternative viral envelope glycoprotein; engineered with acell type-specific receptor (e.g., genetic modification(s) of one ormore viral envelope glycoproteins to incorporate a targeting ligand suchas a peptide ligand, a single chain antibody, or a growth factor);and/or engineered to have a molecular bridge with dual specificitieswith one end recognizing a viral glycoprotein and the other endrecognizing a moiety of the target cell surface (e.g., aligand-receptor, monoclonal antibody, avidin-biotin and chemicalconjugation).

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya recombinant retrovirus. In some embodiments, the retrovirus (e.g.,Moloney murine leukemia virus) comprises a reverse transcriptase, e.g.,that allows integration into the host genome. In some embodiments, theretrovirus is replication-competent. In other embodiments, theretrovirus is replication-defective, e.g., having one of more codingregions for the genes necessary for additional rounds of virionreplication and packaging replaced with other genes, or deleted. In someembodiments, the Cas9- and/or gRNA-encoding DNA is delivered by arecombinant lentivirus. For example, the lentivirus isreplication-defective, e.g., does not comprise one or more genesrequired for viral replication.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya recombinant adenovirus. In some embodiments, the adenovirus isengineered to have reduced immunity in human.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya recombinant AAV. In some embodiments, the AAV does not incorporate itsgenome into that of a host cell, e.g., a target cell as describe herein.In some embodiments, the AAV can incorporate at least part of its genomeinto that of a host cell, e.g., a target cell as described herein. Insome embodiments, the AAV is a self-complementary adeno-associated virus(scAAV), e.g., a scAAV that packages both strands which anneal togetherto form double stranded DNA. AAV serotypes that may be used in thedisclosed methods, include AAV1, AAV2, modified AAV2 (e.g.,modifications at Y444F, Y500F, Y730F and/or S662V), AAV3, modified AAV3(e.g., modifications at Y705F, Y731F and/or T492V), AAV4, AAV5, AAV6,modified AAV6 (e.g., modifications at S663V and/or T492V), AAV8, AAV8.2, AAV9, AAV rh10, and pseudotyped AAV, such as AAV2/8, AAV2/5 andAAV2/6 can also be used in the disclosed methods. In an embodiment, anAAV capsid that can be used in the methods described herein is a capsidsequence from serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,AAV9, AAV.rh8, AAV.rh10, AAV.rh32/33, AAV.rh43, AAV.rh64R1, or AAV7m8.Exemplary AAV serotypes and ITR sequences are disclosed in Table 25.

In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered in are-engineered AAV capsid, e.g., with 50% or greater, e.g., 60% orgreater, 70% or greater, 80% or greater, 90% or greater, or 95% orgreater, sequence homology with a capsid sequence from serotypes AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh8, AAV.rh10,AAV.rh32/33, AAV.rh43, or AAV.rh64R1.

In an embodiment, the Cas9- and/or gRNA-encoding DNA is delivered by achimeric AAV capsid. Exemplary chimeric AAV capsids include, but are notlimited to, AAV9i1, AAV2i8, AAV-DJ, AAV2G9, AAV2i8G9, or AAV8G9.

In an embodiment, the AAV is a self-complementary adeno-associated virus(scAAV), e.g., a scAAV that packages both strands which anneal togetherto form double stranded DNA.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya hybrid virus, e.g., a hybrid of one or more of the viruses describedherein. In an embodiment, the hybrid virus is hybrid of an AAV (e.g., ofany AAV serotype), with a Bocavirus, B19 virus, porcine AAV, goose AAV,feline AAV, canine AAV, or MVM.

A packaging cell is used to form a virus particle that is capable ofinfecting a target cell. Such a cell includes a 293 cell, which canpackage adenovirus, and a ψ2 cell or a PA317 cell, which can packageretrovirus. A viral vector used in gene therapy is usually generated bya producer cell line that packages a nucleic acid vector into a viralparticle. The vector typically contains the minimal viral sequencesrequired for packaging and subsequent integration into a host or targetcell (if applicable), with other viral sequences being replaced by anexpression cassette encoding the protein to be expressed, e.g., Cas9.For example, an AAV vector used in gene therapy typically only possessesinverted terminal repeat (ITR) sequences from the AAV genome which arerequired for packaging and gene expression in the host or target cell.The missing viral functions can be supplied in trans by the packagingcell line and/or plasmid containing E2A, E4, and VA genes fromadenovirus, and plasmid encoding Rep and Cap genes from AAV, asdescribed in “Triple Transfection Protocol.” Henceforth, the viral DNAis packaged in a cell line, which contains a helper plasmid encoding theother AAV genes, namely rep and cap, but lacking ITR sequences. Inembodiment, the viral DNA is packaged in a producer cell line, whichcontains E1A and/or E1B genes from adenovirus. The cell line is alsoinfected with adenovirus as a helper. The helper virus (e.g., adenovirusor HSV) or helper plasmid promotes replication of the AAV vector andexpression of AAV genes from the plasmid with ITRs. The helper plasmidis not packaged in significant amounts due to a lack of ITR sequences.Contamination with adenovirus can be reduced by, e.g., heat treatment towhich adenovirus is more sensitive than AAV.

In an embodiment, the viral vector has the ability of cell type and/ortissue type recognition. For example, the viral vector can bepseudotyped with a different/alternative viral envelope glycoprotein;engineered with a cell type-specific receptor (e.g., geneticmodification of the viral envelope glycoproteins to incorporatetargeting ligands such as a peptide ligand, a single chain antibody, agrowth factor); and/or engineered to have a molecular bridge with dualspecificities with one end recognizing a viral glycoprotein and theother end recognizing a moiety of the target cell surface (e.g.,ligand-receptor, monoclonal antibody, avidin-biotin and chemicalconjugation).

In an embodiment, the viral vector achieves cell type specificexpression. For example, a tissue-specific promoter can be constructedto restrict expression of the transgene (Cas 9 and gRNA) in only thetarget cell. The specificity of the vector can also be mediated bymicroRNA-dependent control of transgene expression. In an embodiment,the viral vector has increased efficiency of fusion of the viral vectorand a target cell membrane. For example, a fusion protein such asfusion-competent hemagglutin (HA) can be incorporated to increase viraluptake into cells. In an embodiment, the viral vector has the ability ofnuclear localization. For example, a virus that requires the breakdownof the cell wall (during cell division) and therefore will not infect anon-diving cell can be altered to incorporate a nuclear localizationpeptide in the matrix protein of the virus thereby enabling thetransduction of non-proliferating cells.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya non-vector based method (e.g., using naked DNA or DNA complexes). Forexample, the DNA can be delivered, e.g., by organically modified silicaor silicate (Ormosil), electroporation, gene gun, sonoporation,magnetofection, lipid-mediated transfection, dendrimers, inorganicnanoparticles, calcium phosphates, or a combination thereof.

In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered bya combination of a vector and a non-vector based method. For example, avirosome comprises a liposome combined with an inactivated virus (e.g.,HIV or influenza virus), which can result in more efficient genetransfer, e.g., in a respiratory epithelial cell than either a viral ora liposomal method alone.

In an embodiment, the delivery vehicle is a non-viral vector. In anembodiment, the non-viral vector is an inorganic nanoparticle. Exemplaryinorganic nanoparticles include, e.g., magnetic nanoparticles (e.g.,Fe₃MnO₂) and silica. The outer surface of the nanoparticle can beconjugated with a positively charged polymer (e.g., polyethylenimine,polylysine, polyserine) which allows for attachment (e.g., conjugationor entrapment) of payload. In an embodiment, the non-viral vector is anorganic nanoparticle (e.g., entrapment of the payload inside thenanoparticle). Exemplary organic nanoparticles include, e.g., SNALPliposomes that contain cationic lipids together with neutral helperlipids which are coated with polyethylene glycol (PEG) and protamine andnucleic acid complex coated with lipid coating.

Exemplary lipids for gene transfer are shown below in Table 21.

TABLE 21 Lipids Used for Gene Transfer Lipid Abbreviation Feature1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine DOPC Helper1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine DOPE HelperCholesterol Helper N-[1-(2,3-Dioleyloxy)prophyl]N,N,N-trimethylammoniumDOTMA Cationic chloride 1,2-Dioleoyloxy-3-trimethylammonium-propaneDOTAP Cationic Dioctadecylamidoglycylspermine DOGS CationicN-(3-Aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1- GAP-DLRIE Cationicpropanaminium bromide Cetyltrimethyl ammonium bromide CTAB Cationic6-Lauroxyhexyl ornithinate LHON Cationic1-(2,3-Dioleoyloxypropyl)-2,4,6-trimethylpyridinium 2Oc Cationic2,3-Dioleyloxy-N-[2(sperminecarboxamido-ethyl]-N,N-dimethyl- DOSPACationic 1-propanaminium trifluoroacetate1,2-Dioleyl-3-trimethylammonium-propane DOPA CationicN-(2-Hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1- MDRIE Cationicpropanaminium bromide Dimyristooxypropyl dimethyl hydroxyethyl ammoniumbromide DMRI Cationic3β-[N-(N',N'-Dimethylaminoethane)-carbamoyl]cholesterol DC-Chol CationicBis-guanidium-tren-cholesterol BGTC Cationic1,3-Diodeoxy-2-(6-carboxy-spermyl)-propylamide DOSPER CationicDimethyloctadecylammonium bromide DDAB CationicDioctadecylamidoglicylspermidin DSL Cationicrac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]- CLIP-1 Cationicdimethylammonium chloride rac-[2(2,3-Dihexadecyloxypropyl- CLIP-6Cationic oxymethyloxy)ethyl]trimethylammonium bromideEthyldimyristoylphosphatidylcholine EDMPC Cationic1,2-Distearyloxy-N,N-dimethyl-3-aminopropane DSDMA Cationic1,2-Dimyristoyl-trimethylammonium propane DMTAP CationicO,O′-Dimyristyl-N-lysyl aspartate DMKE Cationic1,2-Distearoyl-sn-glycero-3-ethylphosphocholine DSEPC CationicN-Palmitoyl D-erythro-sphingosyl carbamoyl-spermine CCS CationicN-t-Butyl-N0-tetradecyl-3-tetradecylaminopropionamidine diC14-amidineCationic Octadecenolyoxy[ethyl-2-heptadecenyl-3 hydroxyethyl] DOTIMCationic imidazolinium chlorideN1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine CDAN Cationic2-(3-[Bis(3-amino-propyl)-amino]propylamino)-N- RPR209120 Cationicditetradecylcarbamoylme-ethyl-acetamide1,2-dilinoleyloxy-3-dimethylaminopropane DLinDMA Cationic2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane DLin-KC2- CationicDMA dilinoleyl-methyl-4-dimethylaminobutyrate DLin-MC3- Cationic DMAExemplary polymers for gene transfer are shown below in Table 22.

TABLE 22 Polymers Used for Gene Transfer Polymer AbbreviationPoly(ethylene)glycol PEG Polyethylenimine PEIDithiobis(succinimidylpropionate) DSPDimethyl-3,3′-dithiobispropionimidate DTBP Poly(ethyleneimine)biscarbamate PEIC Poly(L-lysine) PLL Histidine modified PLLPoly(N-vinylpyrrolidone) PVP Poly(propylenimine) PPI Poly(amidoamine)PAMAM Poly(amidoethylenimine) SS-PAEI Triethylenetetramine TETAPoly(β-aminoester) Poly(4-hydroxy-L-proline ester) PHP Poly(allylamine)Poly(α-[4-aminobutyl]-L-glycolic acid) PAGA Poly(D,L-lactic-co-glycolicacid) PLGA Poly(N-ethyl-4-vinylpyridinium bromide) Poly(phosphazene)sPPZ Poly(phosphoester)s PPE Poly(phosphoramidate)s PPAPoly(N-2-hydroxypropylmethacrylamide) pHPMA Poly(2-(dimethylamino)ethylmethacrylate) pDMAEMA Poly(2-aminoethyl propylene phosphate) PPE-EAChitosan Galactosylated chitosan N-Dodacylated chitosan Histone CollagenDextran-spermine D-SPM

In an embodiment, the vehicle has targeting modifications to increasetarget cell update of nanoparticles and liposomes, e.g., cell specificantigens, monoclonal antibodies, single chain antibodies, aptamers,polymers, sugars, and cell penetrating peptides. In an embodiment, thevehicle uses fusogenic and endosome-destabilizing peptides/polymers. Inan embodiment, the vehicle undergoes acid-triggered conformationalchanges (e.g., to accelerate endosomal escape of the cargo). In anembodiment, a stimuli-cleavable polymer is used, e.g., for release in acellular compartment. For example, disulfide-based cationic polymersthat are cleaved in the reducing cellular environment can be used.

In an embodiment, the delivery vehicle is a biological non-viraldelivery vehicle. In an embodiment, the vehicle is an attenuatedbacterium (e.g., naturally or artificially engineered to be invasive butattenuated to prevent pathogenesis and expressing the transgene (e.g.,Listeria monocytogenes, certain Salmonella strains, Bifidobacteriumlongum, and modified Escherichia coli), bacteria having nutritional andtissue-specific tropism to target specific tissues, bacteria havingmodified surface proteins to alter target tissue specificity). In anembodiment, the vehicle is a genetically modified bacteriophage (e.g.,engineered phages having large packaging capacity, less immunogenic,containing mammalian plasmid maintenance sequences and havingincorporated targeting ligands). In an embodiment, the vehicle is amammalian virus-like particle. For example, modified viral particles canbe generated (e.g., by purification of the “empty” particles followed byex vivo assembly of the virus with the desired cargo). The vehicle canalso be engineered to incorporate targeting ligands to alter targettissue specificity. In an embodiment, the vehicle is a biologicalliposome. For example, the biological liposome is a phospholipid-basedparticle derived from human cells (e.g., erythrocyte ghosts, which arered blood cells broken down into spherical structures derived from thesubject (e.g., tissue targeting can be achieved by attachment of varioustissue or cell-specific ligands), or secretory exosomes—subject (i.e.,patient) derived membrane-bound nanovesicle (30-100 nm) of endocyticorigin (e.g., can be produced from various cell types and can thereforebe taken up by cells without the need of for targeting ligands).

In an embodiment, one or more nucleic acid molecules (e.g., DNAmolecules) other than the components of a Cas system, e.g., the Cas9molecule component and/or the gRNA molecule component described herein,are delivered. In an embodiment, the nucleic acid molecule is deliveredat the same time as one or more of the components of the Cas system aredelivered. In an embodiment, the nucleic acid molecule is deliveredbefore or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2weeks, or 4 weeks) one or more of the components of the Cas system aredelivered. In an embodiment, the nucleic acid molecule is delivered by adifferent means than one or more of the components of the Cas system,e.g., the Cas9 molecule component and/or the gRNA molecule component,are delivered. The nucleic acid molecule can be delivered by any of thedelivery methods described herein. For example, the nucleic acidmolecule can be delivered by a viral vector, e.g., anintegration-deficient lentivirus, and the Cas9 molecule component and/orthe gRNA molecule component can be delivered by electroporation, e.g.,such that the toxicity caused by nucleic acids (e.g., DNAs) can bereduced. In an embodiment, the nucleic acid molecule encodes atherapeutic protein, e.g., a protein described herein. In an embodiment,the nucleic acid molecule encodes an RNA molecule, e.g., an RNA moleculedescribed herein.

Delivery of RNA Encoding a Cas9 Molecule

RNA encoding Cas9 molecules (e.g., eaCas9 molecules) and/or gRNAmolecules, can be delivered into cells, e.g., target cells describedherein, by art-known methods or as described herein. For example,Cas9-encoding and/or gRNA-encoding RNA can be delivered, e.g., bymicroinjection, electroporation, lipid-mediated transfection,peptide-mediated delivery, or a combination thereof. Cas9-encodingand/or gRNA-encoding RNA can be conjugated to molecules (e.g., GalNAc)promoting uptake by the target cells (e.g., target cells describedherein).

Delivery Cas9 Protein

Cas9 molecules (e.g., eaCas9 molecules) can be delivered into cells byart-known methods or as described herein. For example, Cas9 proteinmolecules can be delivered, e.g., by microinjection, electroporation,lipid-mediated transfection, peptide-mediated delivery, or a combinationthereof. Delivery can be accompanied by DNA encoding a gRNA or by agRNA. Cas9-encoding and/or gRNA-encoding RNA can be conjugated tomolecules (e.g., GalNAc) promoting uptake by the target cells (e.g.,target cells described herein).

Route of Administration

Systemic modes of administration include oral and parenteral routes.Parenteral routes include, by way of example, intravenous, intrarterial,intramuscular, intradermal, subcutaneous, intranasal and intraperitonealroutes. Components administered systemically may be modified orformulated to target the components to the eye.

Local modes of administration include, by way of example, intraocular,intraorbital, subconjuctival, intravitreal, subretinal or transscleralroutes. In an embodiment, significantly smaller amounts of thecomponents (compared with systemic approaches) may exert an effect whenadministered locally (for example, intravitreally) compared to whenadministered systemically (for example, intravenously). Local modes ofadministration can reduce or eliminate the incidence of potentiallytoxic side effects that may occur when therapeutically effective amountsof a component are administered systemically.

In an embodiment, components described herein are deliveredsubretinally, e.g., by subretinal injection. Subretinal injections maybe made directly into the macular, e.g., submacular injection.

In an embodiment, components described herein are delivered byintravitreal injection. Intravitreal injection has a relatively low riskof retinal detachment. In an embodiment, nanoparticle or viral, e.g.,AAV vector, is delivered intravitreally.

Methods for administration of agents to the eye are known in the medicalarts and can be used to administer components described herein.Exemplary methods include intraocular injection (e.g., retrobulbar,subretinal, submacular, intravitreal and intrachoridal), iontophoresis,eye drops, and intraocular implantation (e.g., intravitreal, sub-Tenonsand sub-conjunctival).

Administration may be provided as a periodic bolus (for example,subretinally, intravenously or intravitreally) or as continuous infusionfrom an internal reservoir (for example, from an implant disposed at anintra- or extra-ocular location (see, U.S. Pat. Nos. 5,443,505 and5,766,242)) or from an external reservoir (for example, from anintravenous bag). Components may be administered locally, for example,by continuous release from a sustained release drug delivery deviceimmobilized to an inner wall of the eye or via targeted transscleralcontrolled release into the choroid (see, e.g., PCT/US00/00207;PCT/US02/14279; Ambati 2000a; Ambati 2000b. A variety of devicessuitable for administering components locally to the inside of the eyeare known in the art. See, for example, U.S. Pat. Nos. 6,251,090;6,299,895; 6,416,777; and 6,413,540; and PCT Appl. No. PCT/US00/28187.

In addition, components may be formulated to permit release over aprolonged period of time. A release system can include a matrix of abiodegradable material or a material which releases the incorporatedcomponents by diffusion. The components can be homogeneously orheterogeneously distributed within the release system. A variety ofrelease systems may be useful, however, the choice of the appropriatesystem will depend upon rate of release required by a particularapplication. Both non-degradable and degradable release systems can beused. Suitable release systems include polymers and polymeric matrices,non-polymeric matrices, or inorganic and organic excipients and diluentssuch as, but not limited to, calcium carbonate and sugar (for example,trehalose). Release systems may be natural or synthetic. However,synthetic release systems are preferred because generally they are morereliable, more reproducible and produce more defined release profiles.The release system material can be selected so that components havingdifferent molecular weights are released by diffusion through ordegradation of the material.

Representative synthetic, biodegradable polymers include, for example:polyamides such as poly(amino acids) and poly(peptides); polyesters suchas poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolicacid), and poly(caprolactone); poly(anhydrides); polyorthoesters;polycarbonates; and chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), copolymers and mixtures thereof.Representative synthetic, non-degradable polymers include, for example:polyethers such as poly(ethylene oxide), poly(ethylene glycol), andpoly(tetramethylene oxide); vinyl polymers-polyacrylates andpolymethacrylates such as methyl, ethyl, other alkyl, hydroxyethylmethacrylate, acrylic and methacrylic acids, and others such aspoly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate);poly(urethanes); cellulose and its derivatives such as alkyl,hydroxyalkyl, ethers, esters, nitrocellulose, and various celluloseacetates; polysiloxanes; and any chemical derivatives thereof(substitutions, additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art), copolymers and mixtures thereof.

Poly(lactide-co-glycolide) microsphere can also be used for intraocularinjection. Typically the microspheres are composed of a polymer oflactic acid and glycolic acid, which are structured to form hollowspheres. The spheres can be approximately 15-30 microns in diameter andcan be loaded with components described herein.

Bi-Modal or Differential Delivery of Components

Separate delivery of the components of a Cas system, e.g., the Cas9molecule component and the gRNA molecule component, and moreparticularly, delivery of the components by differing modes, can enhanceperformance, e.g., by improving tissue specificity and safety.

In an embodiment, the Cas9 molecule and the gRNA molecule are deliveredby different modes, or as sometimes referred to herein as differentialmodes. Different or differential modes, as used herein, refer modes ofdelivery that confer different pharmacodynamic or pharmacokineticproperties on the subject component molecule, e.g., a Cas9 molecule,gRNA molecule, template nucleic acid, or payload. For example, the modesof delivery can result in different tissue distribution, differenthalf-life, or different temporal distribution, e.g., in a selectedcompartment, tissue, or organ.

Some modes of delivery, e.g., delivery by a nucleic acid vector thatpersists in a cell, or in progeny of a cell, e.g., by autonomousreplication or insertion into cellular nucleic acid, result in morepersistent expression of and presence of a component. Examples includeviral, e.g., adeno associated virus or lentivirus, delivery.

By way of example, the components, e.g., a Cas9 molecule and a gRNAmolecule, can be delivered by modes that differ in terms of resultinghalf-life or persistent of the delivered component the body, or in aparticular compartment, tissue or organ. In an embodiment, a gRNAmolecule can be delivered by such modes. The Cas9 molecule component canbe delivered by a mode which results in less persistence or lessexposure to the body or a particular compartment or tissue or organ.

More generally, in an embodiment, a first mode of delivery is used todeliver a first component and a second mode of delivery is used todeliver a second component. The first mode of delivery confers a firstpharmacodynamic or pharmacokinetic property. The first pharmacodynamicproperty can be, e.g., distribution, persistence, or exposure, of thecomponent, or of a nucleic acid that encodes the component, in the body,a compartment, tissue or organ. The second mode of delivery confers asecond pharmacodynamic or pharmacokinetic property. The secondpharmacodynamic property can be, e.g., distribution, persistence, orexposure, of the component, or of a nucleic acid that encodes thecomponent, in the body, a compartment, tissue or organ.

In an embodiment, the first pharmacodynamic or pharmacokinetic property,e.g., distribution, persistence or exposure, is more limited than thesecond pharmacodynamic or pharmacokinetic property.

In an embodiment, the first mode of delivery is selected to optimize,e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g.,distribution, persistence or exposure.

In an embodiment, the second mode of delivery is selected to optimize,e.g., maximize, a pharmacodynamic or pharmacokinetic property, e.g.,distribution, persistence or exposure.

In an embodiment, the first mode of delivery comprises the use of arelatively persistent element, e.g., a nucleic acid, e.g., a plasmid orviral vector, e.g., an AAV or lentivirus. As such vectors are relativelypersistent product transcribed from them would be relatively persistent.

In an embodiment, the second mode of delivery comprises a relativelytransient element, e.g., an RNA or protein.

In an embodiment, the first component comprises gRNA, and the deliverymode is relatively persistent, e.g., the gRNA is transcribed from aplasmid or viral vector, e.g., an AAV or lentivirus. Transcription ofthese genes would be of little physiological consequence because thegenes do not encode for a protein product, and the gRNAs are incapableof acting in isolation. The second component, a Cas9 molecule, isdelivered in a transient manner, for example as mRNA or as protein,ensuring that the full Cas9 molecule/gRNA molecule complex is onlypresent and active for a short period of time.

Furthermore, the components can be delivered in different molecular formor with different delivery vectors that complement one another toenhance safety and tissue specificity.

Use of differential delivery modes can enhance performance, safety andefficacy. E.g., the likelihood of an eventual off-target modificationcan be reduced. Delivery of immunogenic components, e.g., Cas9molecules, by less persistent modes can reduce immunogenicity, aspeptides from the bacterially-derived Cas enzyme are displayed on thesurface of the cell by MHC molecules. A two-part delivery system canalleviate these drawbacks.

Differential delivery modes can be used to deliver components todifferent, but overlapping target regions. The formation active complexis minimized outside the overlap of the target regions. Thus, in anembodiment, a first component, e.g., a gRNA molecule is delivered by afirst delivery mode that results in a first spatial, e.g., tissue,distribution. A second component, e.g., a Cas9 molecule is delivered bya second delivery mode that results in a second spatial, e.g., tissue,distribution. In an embodiment the first mode comprises a first elementselected from a liposome, nanoparticle, e.g., polymeric nanoparticle,and a nucleic acid, e.g., viral vector. The second mode comprises asecond element selected from the group. In an embodiment, the first modeof delivery comprises a first targeting element, e.g., a cell specificreceptor or an antibody, and the second mode of delivery does notinclude that element. In embodiment, the second mode of deliverycomprises a second targeting element, e.g., a second cell specificreceptor or second antibody.

When the Cas9 molecule is delivered in a virus delivery vector, aliposome, or polymeric nanoparticle, there is the potential for deliveryto and therapeutic activity in multiple tissues, when it may bedesirable to only target a single tissue. A two-part delivery system canresolve this challenge and enhance tissue specificity. If the gRNAmolecule and the Cas9 molecule are packaged in separated deliveryvehicles with distinct but overlapping tissue tropism, the fullyfunctional complex is only be formed in the tissue that is targeted byboth vectors.

Ex Vivo Delivery

In some embodiments, components described in Table 18 are introducedinto cells which are then introduced into the subject. Methods ofintroducing the components can include, e.g., any of the deliverymethods described in Table 19.

VIII. Modified Nucleosides, Nucleotides, and Nucleic Acids

Modified nucleosides and modified nucleotides can be present in nucleicacids, e.g., particularly gRNA, but also other forms of RNA, e.g., mRNA,RNAi, or siRNA. As described herein, “nucleoside” is defined as acompound containing a five-carbon sugar molecule (a pentose or ribose)or derivative thereof, and an organic base, purine or pyrimidine, or aderivative thereof. As described herein, “nucleotide” is defined as anucleoside further comprising a phosphate group.

Modified nucleosides and nucleotides can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linkingphosphate oxygens and/or of one or more of the linking phosphate oxygensin the phosphodiester backbone linkage;

(ii) alteration, e.g., replacement, of a constituent of the ribosesugar, e.g., of the 2′ hydroxyl on the ribose sugar;

(iii) wholesale replacement of the phosphate moiety with “dephospho”linkers;

(iv) modification or replacement of a naturally occurring nucleobase;

(v) replacement or modification of the ribose-phosphate backbone;

(vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g.,removal, modification or replacement of a terminal phosphate group orconjugation of a moiety; and

(vii) modification of the sugar.

The modifications listed above can be combined to provide modifiednucleosides and nucleotides that can have two, three, four, or moremodifications. For example, a modified nucleoside or nucleotide can havea modified sugar and a modified nucleobase. In an embodiment, every baseof a gRNA is modified, e.g., all bases have a modified phosphate group,e.g., all are phosphorothioate groups. In an embodiment, all, orsubstantially all, of the phosphate groups of a unimolecular or modulargRNA molecule are replaced with phosphorothioate groups.

In an embodiment, modified nucleotides, e.g., nucleotides havingmodifications as described herein, can be incorporated into a nucleicacid, e.g., a “modified nucleic acid.” In some embodiments, the modifiednucleic acids comprise one, two, three or more modified nucleotides. Insome embodiments, at least 5% (e.g., at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about80%, at least about 85%, at least about 90%, at least about 95%, orabout 100%) of the positions in a modified nucleic acid are a modifiednucleotides.

Unmodified nucleic acids can be prone to degradation by, e.g., cellularnucleases. For example, nucleases can hydrolyze nucleic acidphosphodiester bonds. Accordingly, in one aspect the modified nucleicacids described herein can contain one or more modified nucleosides ornucleotides, e.g., to introduce stability toward nucleases.

In some embodiments, the modified nucleosides, modified nucleotides, andmodified nucleic acids described herein can exhibit a reduced innateimmune response when introduced into a population of cells, both in vivoand ex vivo. The term “innate immune response” includes a cellularresponse to exogenous nucleic acids, including single stranded nucleicacids, generally of viral or bacterial origin, which involves theinduction of cytokine expression and release, particularly theinterferons, and cell death. In some embodiments, the modifiednucleosides, modified nucleotides, and modified nucleic acids describedherein can disrupt binding of a major groove interacting partner withthe nucleic acid. In some embodiments, the modified nucleosides,modified nucleotides, and modified nucleic acids described herein canexhibit a reduced innate immune response when introduced into apopulation of cells, both in vivo and ex vivo, and also disrupt bindingof a major groove interacting partner with the nucleic acid.

Definitions of Chemical Groups

As used herein, “alkyl” is meant to refer to a saturated hydrocarbongroup which is straight-chained or branched. Example alkyl groupsinclude methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl),butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl,isopentyl, neopentyl), and the like. An alkyl group can contain from 1to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8,from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

As used herein, “aryl” refers to monocyclic or polycyclic (e.g., having2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example,phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and thelike. In some embodiments, aryl groups have from 6 to about 20 carbonatoms.

As used herein, “alkenyl” refers to an aliphatic group containing atleast one double bond.

As used herein, “alkynyl” refers to a straight or branched hydrocarbonchain containing 2-12 carbon atoms and characterized in having one ormore triple bonds. Examples of alkynyl groups include, but are notlimited to, ethynyl, propargyl, and 3-hexynyl.

As used herein, “arylalkyl” or “aralkyl” refers to an alkyl moiety inwhich an alkyl hydrogen atom is replaced by an aryl group. Aralkylincludes groups in which more than one hydrogen atom has been replacedby an aryl group. Examples of“arylalkyl” or “aralkyl” include benzyl,2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and tritylgroups.

As used herein, “cycloalkyl” refers to a cyclic, bicyclic, tricyclic, orpolycyclic non-aromatic hydrocarbon groups having 3 to 12 carbons.Examples of cycloalkyl moieties include, but are not limited to,cyclopropyl, cyclopentyl, and cyclohexyl.

As used herein, “heterocyclyl” refers to a monovalent radical of aheterocyclic ring system. Representative heterocyclyls include, withoutlimitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl,pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl,dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.

As used herein, “heteroaryl” refers to a monovalent radical of aheteroaromatic ring system. Examples of heteroaryl moieties include, butare not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl,pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl,pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl,quinolyl, and pteridinyl.

Phosphate Backbone Modifications

Phosphate Group

In some embodiments, the phosphate group of a modified nucleotide can bemodified by replacing one or more of the oxygens with a differentsubstituent. Further, the modified nucleotide, e.g., modified nucleotidepresent in a modified nucleic acid, can include the wholesalereplacement of an unmodified phosphate moiety with a modified phosphateas described herein. In some embodiments, the modification of thephosphate backbone can include alterations that result in either anuncharged linker or a charged linker with unsymmetrical chargedistribution.

Examples of modified phosphate groups include, phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. In some embodiments, one of the non-bridging phosphateoxygen atoms in the phosphate backbone moiety can be replaced by any ofthe following groups: sulfur (S), selenium (Se), BR₃ (wherein R can be,e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group,and the like), H, NR₂ (wherein R can be, e.g., hydrogen, alkyl, oraryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorousatom in an unmodified phosphate group is achiral. However, replacementof one of the non-bridging oxygens with one of the above atoms or groupsof atoms can render the phosphorous atom chiral; that is to say that aphosphorous atom in a phosphate group modified in this way is astereogenic center. The stereogenic phosphorous atom can possess eitherthe “R” configuration (herein Rp) or the “S” configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur.The phosphorus center in the phosphorodithioates is achiral whichprecludes the formation of oligoribonucleotide diastereomers. In someembodiments, modifications to one or both non-bridging oxygens can alsoinclude the replacement of the non-bridging oxygens with a groupindependently selected from S, Se, B, C, H, N, and OR (R can be, e.g.,alkyl or aryl).

The phosphate linker can also be modified by replacement of a bridgingoxygen, (i.e., the oxygen that links the phosphate to the nucleoside),with nitrogen (bridged phosphoroamidates), sulfur (bridgedphosphorothioates) and carbon (bridged methylenephosphonates). Thereplacement can occur at either linking oxygen or at both of the linkingoxygens.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors. In some embodiments, the charge phosphate group can bereplaced by a neutral moiety.

Examples of moieties which can replace the phosphate group can include,without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane,carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxidelinker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime,methyleneimino, methylenemethylimino, methylenehydrazo,methylenedimethylhydrazo and methyleneoxymethylimino.

Replacement of the Ribophosphate Backbone

Scaffolds that can mimic nucleic acids can also be constructed whereinthe phosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. In some embodiments, thenucleobases can be tethered by a surrogate backbone. Examples caninclude, without limitation, the morpholino, cyclobutyl, pyrrolidine andpeptide nucleic acid (PNA) nucleoside surrogates.

Sugar Modifications

The modified nucleosides and modified nucleotides can include one ormore modifications to the sugar group. For example, the 2′ hydroxylgroup (OH) can be modified or replaced with a number of different “oxy”or “deoxy” substituents. In some embodiments, modifications to the 2′hydroxyl group can enhance the stability of the nucleic acid since thehydroxyl can no longer be deprotonated to form a 2′-alkoxide ion. The2′-alkoxide can catalyze degradation by intramolecular nucleophilicattack on the linker phosphorus atom.

Examples of “oxy”-2′ hydroxyl group modifications can include alkoxy oraryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or a sugar); polyethyleneglycols (PEG),O(CH₂CH₂O)_(n)CH₂CH₂OR wherein R can be, e.g., H or optionallysubstituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8,from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4to 16, and from 4 to 20). In some embodiments, the “oxy”-2′ hydroxylgroup modification can include “locked” nucleic acids (LNA) in which the2′ hydroxyl can be connected, e.g., by a C₁₋₆ alkylene or C1-6heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, whereexemplary bridges can include methylene, propylene, ether, or aminobridges; O-amino (wherein amino can be, e.g., NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy,O(CH₂)_(n)-amino, (wherein amino can be, e.g., NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroarylamino, ethylenediamine, or polyamino). In some embodiments,the “oxy”-2′ hydroxyl group modification can include the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, e.g., a PEG derivative).

“Deoxy” modifications can include hydrogen (i.e. deoxyribose sugars,e.g., at the overhang portions of partially ds RNA); halo (e.g., bromo,chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino,heteroarylamino, diheteroarylamino, or amino acid);NH(CH₂CH₂NH)_(n)CH₂CH₂-amino (wherein amino can be, e.g., as describedherein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl,aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which maybe optionally substituted with e.g., an amino as described herein.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified nucleic acid can include nucleotidescontaining e.g., arabinose, as the sugar. The nucleotide “monomer” canhave an alpha linkage at the 1′ position on the sugar, e.g.,alpha-nucleosides. The modified nucleic acids can also include “abasic”sugars, which lack a nucleobase at C-1′. These abasic sugars can also befurther modified at one or more of the constituent sugar atoms. Themodified nucleic acids can also include one or more sugars that are inthe L form, e.g. L-nucleosides.

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary modified nucleosides and modifiednucleotides can include, without limitation, replacement of the oxygenin ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as,e.g., methylene or ethylene); addition of a double bond (e.g., toreplace ribose with cyclopentenyl or cyclohexenyl); ring contraction ofribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ringexpansion of ribose (e.g., to form a 6- or 7-membered ring having anadditional carbon or heteroatom, such as for example, anhydrohexitol,altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that alsohas a phosphoramidate backbone). In some embodiments, the modifiednucleotides can include multicyclic forms (e.g., tricyclo; and“unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA orS-GNA, where ribose is replaced by glycol units attached tophosphodiester bonds), threose nucleic acid (TNA, where ribose isreplaced with α-L-threofuranosyl-(3′→2′)).

Modifications on the Nucleobase

The modified nucleosides and modified nucleotides described herein,which can be incorporated into a modified nucleic acid, can include amodified nucleobase. Examples of nucleobases include, but are notlimited to, adenine (A), guanine (G), cytosine (C), and uracil (U).These nucleobases can be modified or wholly replaced to provide modifiednucleosides and modified nucleotides that can be incorporated intomodified nucleic acids. The nucleobase of the nucleotide can beindependently selected from a purine, a pyrimidine, a purine orpyrimidine analog. In some embodiments, the nucleobase can include, forexample, naturally-occurring and synthetic derivatives of a base.

Uracil

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includewithout limitation pseudouridine (u), pyridin-4-one ribonucleoside,5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s2U),5-aminomethyl-2-thio-uridine (nm⁵s2U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s2U),5-methylaminomethyl-2-seleno-uridine (mnm⁵ se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethyl aminomethyl-2-thio-uridine (cmnm⁵s2U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τcm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm⁵ s2U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹),5-methyl-2-thio-uridine (m⁵ s2U), 1-methyl-4-thio-pseudouridine(m¹s4ψr), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψr),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s2U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s2Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm ⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm ⁵Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm ⁵Um), 3,2′-O-dimethyl-uridine(m³Um), 5-(isopentenyl aminomethyl)-2′-O-methyl-uridine (inm ⁵Um),1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine,2′-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine,5-[3-(1-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine,and hypoxanthine.

Cytosine

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine includewithout limitation 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine,3-methyl-cytidine (m³C), N4-acetyl-cytidine (act), 5-formyl-cytidine(f⁵C), N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C),5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine(hm⁵C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine,pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C),2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k²C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f ⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

Adenine

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine includewithout limitation 2-amino-purine, 2,6-diaminopurine,2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine(e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine,7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms2 m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i6A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N⁶,2′-O-dimethyl-adenosine (m⁶Am), N⁶-Methyl-2′-deoxyadenosine,N6,N6,2′-O-trimethyl-adenosine (m⁶2Am), 1,2′-O-dimethyl-adenosine (mAm), 2′-O-ribosyladenosine (phosphate) (Ar(p)),2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine,2′-F-ara-adenosine, 2′-F-adenosine, 2′-OH-ara-adenosine, andN6-(19-amino-pentaoxanonadecyl)-adenosine.

Guanine

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includewithout limitation inosine (I), 1-methyl-inosine (m¹I), wyosine (imG),methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2),wybutosine (yW), peroxywybutosine (ozyW), hydroxywybutosine (OHyW),undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine(Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ),mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G+),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m′G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m²,7G), N2,N2,7-dimethyl-guanosine (m²,2,7G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-meth thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m′Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m²,7Gm), 2′-O-methyl-inosine (Im),1,2′-O-dimethyl-inosine (m′Im), O⁶-phenyl-2′-deoxyinosine,2′-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine,O⁶-methyl-guanosine, O⁶-Methyl-2′-deoxyguanosine, 2′-F-ara-guanosine,and 2′-F-guanosine.

Modified gRNAs

In some embodiments, the modified nucleic acids can be modified gRNAs.In some embodiments, gRNAs can be modified at the 3′ end. In thisembodiment, the gRNAs can be modified at the 3′ terminal U ribose. Forexample, the two terminal hydroxyl groups of the U ribose can beoxidized to aldehyde groups and a concomitant opening of the ribose ringto afford a modified nucleoside as sown below:

wherein “U” can be an unmodified or modified uridine.

In another embodiment, the 3′ terminal U can be modified with a 2′3′cyclic phosphate as shown below:

wherein “U” can be an unmodified or modified uridine.

In some embodiments, the gRNA molecules may contain 3′ nucleotides whichcan be stabilized against degradation, e.g., by incorporating one ormore of the modified nucleotides described herein. In this embodiment,e.g., uridines can be replaced with modified uridines, e.g.,5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of themodified uridines described herein; adenosines and guanosines can bereplaced with modified adenosines and guanosines, e.g., withmodifications at the 8-position, e.g., 8-bromo guanosine, or with any ofthe modified adenosines or guanosines described herein. In someembodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can beincorporated into the gRNA. In some embodiments, O- and N-alkylatednucleotides, e.g., N6-methyl adenosine, can be incorporated into thegRNA. In some embodiments, sugar-modified ribonucleotides can beincorporated, e.g., wherein the 2′ OH-group is replaced by a groupselected from H, —OR, —R (wherein R can be, e.g., alkyl, cycloalkyl,aryl, aralkyl, heteroaryl or sugar), halo, —SH, —SR (wherein R can be,e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino(wherein amino can be, e.g., NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diarylamino, heteroarylamino,diheteroarylamino, or amino acid); or cyano (—CN). In some embodiments,the phosphate backbone can be modified as described herein, e.g., with aphosphothioate group. In some embodiments, the nucleotides in theoverhang region of the gRNA can each independently be a modified orunmodified nucleotide including, but not limited to 2′-sugar modified,such as, 2-F 2′-O-methyl, thymidine (T),2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine(Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinationsthereof.

In an embodiment, one or more or all of the nucleotides in singlestranded RNA molecule, e.g., a gRNA molecule, are deoxynucleotides.

miRNA Binding Sites

microRNAs (or miRNAs) are naturally occurring cellular 19-25 nucleotidelong noncoding RNAs. They bind to nucleic acid molecules having anappropriate miRNA binding site, e.g., in the 3′ UTR of an mRNA, anddown-regulate gene expression. While not wishing to be bound by theory,in an embodiment, it is believed that the down regulation is either byreducing nucleic acid molecule stability or by inhibiting translation.An RNA species disclosed herein, e.g., an mRNA encoding Cas9 cancomprise an miRNA binding site, e.g., in its 3′UTR. The miRNA bindingsite can be selected to promote down regulation of expression is aselected cell type. By way of example, the incorporation of a bindingsite for miR-122, a microRNA abundant in liver, can inhibit theexpression of the gene of interest in the liver.

Governing gRNA Molecules and the Use Thereof to Limit the Activity of aCas9 System

Methods and compositions that use, or include, a nucleic acid, e.g.,DNA, that encodes a Cas9 molecule or a gRNA molecule, can, in addition,use or include a “governing gRNA molecule.” The governing gRNA can limitthe activity of the other CRISPR/Cas components introduced into a cellor subject. In an embodiment, a gRNA molecule comprises a targetingdomain that is complementary to a target domain on a nucleic acid thatcomprises a sequence that encodes a component of the CRISPR/Cas systemthat is introduced into a cell or subject. In an embodiment, a governinggRNA molecule comprises a targeting domain that is complementary with atarget sequence on: (a) a nucleic acid that encodes a Cas9 molecule; (b)a nucleic acid that encodes a gRNA which comprises a targeting domainthat targets the CEP290 gene (a target gene gRNA); or on more than onenucleic acid that encodes a CRISPR/Cas component, e.g., both (a) and(b). The governing gRNA molecule can complex with the Cas9 molecule toinactivate a component of the system. In an embodiment, a Cas9molecule/governing gRNA molecule complex inactivates a nucleic acid thatcomprises the sequence encoding the Cas9 molecule. In an embodiment, aCas9 molecule/governing gRNA molecule complex inactivates the nucleicacid that comprises the sequence encoding a target gene gRNA molecule.In an embodiment, a Cas9 molecule/governing gRNA molecule complex placestemporal, level of expression, or other limits, on activity of the Cas9molecule/target gene gRNA molecule complex. In an embodiment, a Cas9molecule/governing gRNA molecule complex reduces off-target or otherunwanted activity. In an embodiment, a governing gRNA molecule targetsthe coding sequence, or a control region, e.g., a promoter, for theCRISPR/Cas system component to be negatively regulated. For example, agoverning gRNA can target the coding sequence for a Cas9 molecule, or acontrol region, e.g., a promoter, that regulates the expression of theCas9 molecule coding sequence, or a sequence disposed between the two.In an embodiment, a governing gRNA molecule targets the coding sequence,or a control region, e.g., a promoter, for a target gene gRNA. In anembodiment, a governing gRNA, e.g., a Cas9-targeting or target genegRNA-targeting, governing gRNA molecule, or a nucleic acid that encodesit, is introduced separately, e.g., later, than is the Cas9 molecule ora nucleic acid that encodes it. For example, a first vector, e.g., aviral vector, e.g., an AAV vector, can introduce nucleic acid encoding aCas9 molecule and one or more target gene gRNA molecules, and a secondvector, e.g., a viral vector, e.g., an AAV vector, can introduce nucleicacid encoding a governing gRNA molecule, e.g., a Cas9-targeting ortarget gene gRNA targeting, gRNA molecule. In an embodiment, the secondvector can be introduced after the first. In other embodiments, agoverning gRNA molecule, e.g., a Cas9-targeting or target gene gRNAtargeting, governing gRNA molecule, or a nucleic acid that encodes it,can be introduced together, e.g., at the same time or in the samevector, with the Cas9 molecule or a nucleic acid that encodes it, but,e.g., under transcriptional control elements, e.g., a promoter or anenhancer, that are activated at a later time, e.g., such that after aperiod of time the transcription of Cas9 is reduced. In an embodiment,the transcriptional control element is activated intrinsically. In anembodiment, the transcriptional element is activated via theintroduction of an external trigger.

Typically a nucleic acid sequence encoding a governing gRNA molecule,e.g., a Cas9-targeting gRNA molecule, is under the control of adifferent control region, e.g., promoter, than is the component itnegatively modulates, e.g., a nucleic acid encoding a Cas9 molecule. Inan embodiment, “different control region” refers to simply not beingunder the control of one control region, e.g., promoter, that isfunctionally coupled to both controlled sequences. In an embodiment,different refers to “different control region” in kind or type ofcontrol region. For example, the sequence encoding a governing gRNAmolecule, e.g., a Cas9-targeting gRNA molecule, is under the control ofa control region, e.g., a promoter, that has a lower level ofexpression, or is expressed later than the sequence which encodes is thecomponent it negatively modulates, e.g., a nucleic acid encoding a Cas9molecule.

By way of example, a sequence that encodes a governing gRNA molecule,e.g., a Cas9-targeting governing gRNA molecule, can be under the controlof a control region (e.g., a promoter) described herein, e.g., human U6small nuclear promoter, or human H1 promoter. In an embodiment, asequence that encodes the component it negatively regulates, e.g., anucleic acid encoding a Cas9 molecule, can be under the control of acontrol region (e.g., a promoter) described herein, e.g., CMV, EF-1a,MSCV, PGK, CAG control promoters.

EXAMPLES

The following Examples are merely illustrative and are not intended tolimit the scope or content of the invention in any way.

Example 1: Cloning and Initial Screening of gRNAs

The suitability of candidate gRNAs can be evaluated as described in thisexample. Although described for a chimeric gRNA, the approach can alsobe used to evaluate modular gRNAs.

Cloning gRNAs into Plasmid Vector

For each gRNA, a pair of overlapping oligonucleotides is designed andobtained. Oligonucleotides are annealed and ligated into a digestedvector backbone containing an upstream U6 promoter and the remainingsequence of a long chimeric gRNA. Plasmid is sequence-verified andprepped to generate sufficient amounts of transfection-quality DNA.Alternate promoters maybe used to drive in vivo transcription (e.g., H1promoter) or for in vitro transcription (e.g., T7 promoter).

Cloning gRNAs in Linear dsDNA Molecule (STITCHR)

For each gRNA, a single oligonucleotide is designed and obtained. The U6promoter and the gRNA scaffold (e.g. including everything except thetargeting domain, e.g., including sequences derived from the crRNA andtracrRNA, e.g., including a first complementarity domain; a linkingdomain; a second complementarity domain; a proximal domain; and a taildomain) are separately PCR amplified and purified as dsDNA molecules.The gRNA-specific oligonucleotide is used in a PCR reaction to stitchtogether the U6 and the gRNA scaffold, linked by the targeting domainspecified in the oligonucleotide. Resulting dsDNA molecule (STITCHRproduct) is purified for transfection. Alternate promoters may be usedto drive in vivo transcription (e.g., H1 promoter) or for in vitrotranscription (e.g., T7 promoter). Any gRNA scaffold may be used tocreate gRNAs compatible with Cas9s from any bacterial species.

Initial gRNA Screen

Each gRNA to be tested is transfected, along with a plasmid expressingCas9 and a small amount of a GFP-expressing plasmid into human cells. Inpreliminary experiments, these cells can be immortalized human celllines such as 293T, K562 or U2OS. Alternatively, primary human cells maybe used. In this case, cells may be relevant to the eventual therapeuticcell target (for example, photoreceptor cells). The use of primary cellssimilar to the potential therapeutic target cell population may provideimportant information on gene targeting rates in the context ofendogenous chromatin and gene expression.

Transfection may be performed using lipid transfection (such asLipofectamine or Fugene) or by electroporation. Following transfection,GFP expression can be determined either by fluorescence microscopy or byflow cytometry to confirm consistent and high levels of transfection.These preliminary transfections can comprise different gRNAs anddifferent targeting approaches (17-mers, 20-mers, nuclease,dual-nickase, etc.) to determine which gRNAs/combinations of gRNAs givethe greatest activity.

Efficiency of cleavage with each gRNA may be assessed by measuringNHEJ-induced indel formation at the target locus by a T7E1-type assay orby sequencing. Alternatively, other mismatch-sensitive enzymes, such asCell/Surveyor nuclease, may also be used.

For the T7E1 assay, PCR amplicons are approximately 500-700 bp with theintended cut site placed asymmetrically in the amplicon. Followingamplification, purification and size-verification of PCR products, DNAis denatured and re-hybridized by heating to 95° C. and then slowlycooling. Hybridized PCR products are then digested with T7 EndonucleaseI (or other mismatch-sensitive enzyme) which recognizes and cleavesnon-perfectly matched DNA. If indels are present in the originaltemplate DNA, when the amplicons are denatured and re-annealed, thisresults in the hybridization of DNA strands harboring different indelsand therefore lead to double-stranded DNA that is not perfectly matched.Digestion products may be visualized by gel electrophoresis or bycapillary electrophoresis. The fraction of DNA that is cleaved (densityof cleavage products divided by the density of cleaved and uncleaved)may be used to estimate a percent NHEJ using the following equation: %NHEJ=(1−(1−fraction cleaved)^(1/2)). The T7E1 assay is sensitive down toabout 2-5% NHEJ.

Sequencing may be used instead of, or in addition to, the T7E1 assay.For Sanger sequencing, purified PCR amplicons are cloned into a plasmidbackbone, transformed, miniprepped and sequenced with a single primer.For large sequencing numbers, Sanger sequencing may be used fordetermining the exact nature of indels after determining the NHEJ rateby T7E1.

Sequencing may also be performed using next generation sequencingtechniques. When using next generation sequencing, amplicons may be300-500 bp with the intended cut site placed asymmetrically. FollowingPCR, next generation sequencing adapters and barcodes (for exampleIllumina multiplex adapters and indexes) may be added to the ends of theamplicon, e.g., for use in high throughput sequencing (for example on anIllumina MiSeq). This method allows for detection of very low NHEJrates.

Example 2: Assessment of Gene Targeting by NHEJ

The gRNAs that induce the greatest levels of NHEJ in initial tests canbe selected for further evaluation of gene targeting efficiency. Forexample, cells may be derived from disease subjects, relevant celllines, and/or animal models and, therefore, harbor the relevantmutation.

Following transfection (usually 2-3 days post-transfection) genomic DNAmay be isolated from a bulk population of transfected cells and PCR maybe used to amplify the target region. Following PCR, gene targetingefficiency to generate the desired mutations (either knockout of atarget gene or removal of a target sequence motif) may be determined bysequencing. For Sanger sequencing, PCR amplicons may be 500-700 bp long.For next generation sequencing, PCR amplicons may be 300-500 bp long. Ifthe goal is to knockout gene function, sequencing may be used to assesswhat percent of alleles have undergone NHEJ-induced indels that resultin a frameshift or large deletion or insertion that would be expected todestroy gene function. If the goal is to remove a specific sequencemotif, sequencing may be used to assess what percent of alleles haveundergone NHEJ-induced deletions that span this sequence.

Example 3: Assessment of Activity of Individual gRNAs Targeting CEP290

Guide RNA were identified using a custom guide RNA design software basedon the public tool cas-offinder (Bae 2014). Each gRNA to be tested wasgenerated as a STITCHR product and co-transfected with a plasmidexpressing either S. aureus Cas9 (pAF003) or S. pyogenes Cas9 (pJDS246)into either HEK293 cells or primary fibroblasts derived from and LCA10patient harboring homozygous IVS26 c.2991+1655A to G mutations(hereafter referred to as IVS26 fibroblasts). The pAF003 plasmid encodesthe S. aureus Cas9, with N-terminal and C-terminal nuclear localizationsignals (NLS) and a C-terminal triple flag tag, driven by a CMVpromoter. The pJDS246 plasmid encodes the S. pyogenes Cas9, with aC-terminal nuclear localization signal (NLS) and a C-terminal tripleflag tag, driven by a CMV promoter. gRNA and Cas9-encoding DNA wasintroduced into cells by either Mirus TransIT-293 transfection reagent(for 293 cells) or by Amaxa nucleofection (for IVS26 fibroblasts).Nucleofection was optimized for transfection of IVS26 fibroblasts usingsolution P2 and various pulse codes and assaying for highest levels ofgene editing and cell viability. Transfection efficiency in both celltypes was assessed by transfecting with GFP and assaying expression byfluorescent microscopy. Three to seven days post-transfection, genomicDNA was isolated from bulk populations of transfected cells and theregion of the CEP290 locus surrounding the target site was PCRamplified. PCR amplicons were then cloned into a plasmid backbone usingthe Zero-Blunt TOPO cloning kit (Life Technologies) and transformed intochemically competent Top10 cells. Bacterial colonies were then culturedand plasmid DNA was isolated and sequenced. Sequencing of PCR productsallowed for the detection and quantification of targeted insertion anddeletion (indel) events at the target site. FIGS. 11A and 11B show therates of indels induced by various gRNAs at the CEP290 locus. FIG. 11Ashows gene editing (% indels) as assessed by sequencing for S. pyogenesand S. aureus gRNAs when co-expressed with Cas9 in patient-derived IVS26primary fibroblasts. FIG. 11B shows gene editing (% indels) as assessedby sequencing for S. aureus gRNAs when co-expressed with Cas9 in HEK293cells.

Example 4: Detection of gRNA Pair-Induced Deletions by PCR

To assess the ability of a pair of gRNAs to induce a genomic deletion(in which the sequence between the two cut sites is removed), PCR wasperformed across the predicted deletion. Pairs of gRNAs (encoded asSTITCHR products) were co-transfected with pAF003 into IVS26fibroblasts. Genomic DNA was isolated from transfected cells and PCR wasperformed to amplify a segment of the CEP290 locus spanning the twopredicted cut sites. PCR was run on a QIAxcel capillary electrophoresismachine. The predicted amplicon on a wildtype allele is 1816 bps.Assuming that cleavage occurs within the gRNA target region, ampliconsizes for alleles having undergone the deletion event were calculatedand the presence of this smaller band indicates that the desired genomicdeletion event has occurred (Table 23).

TABLE 23 Deletion Deletion Amplicon with amplicon Left gRNA Right gRNASize deletion detected?  1 CEP290-367 CEP290-16  590 1226 no  2CEP290-367 CEP290-203 688 1128 no  3 CEP290-367 CEP290-132 815 1001 no 4 CEP290-367 CEP290-139 1265 551 no  5 CEP290-312 CEP290-11  790 1026yes  6 CEP290-312 CEP290-252 973 843 no  7 CEP290-312 CEP290-64  976 840yes  8 CEP290-312 CEP290-230 1409 407 yes  9 CEP290-12  CEP290-11  191797 no 10 CEP290-12  CEP290-252 202 1614 no 11 CEP290-12  CEP290-64 205 1611 no 12 CEP290-12  CEP290-230 638 1178 no 13 CEP290-17 CEP290-16  19 1797 no 14 CEP290-17  CEP290-203 117 1699 no 15 CEP290-17 CEP290-132 244 1572 no 16 CEP290-17  CEP290-139 693 1123 no 17CEP290-374 CEP290-16  799 1017 no 18 CEP290-374 CEP290-203 897 919 no 19CEP290-374 CEP290-132 1024 792 no 20 CEP290-374 CEP290-139 1473 343 no21 CEP290-368 CEP290-16  854 962 no 22 CEP290-368 CEP290-203 952 864 no23 CEP290-368 CEP290-132 1079 737 no 24 CEP290-368 CEP290-139 1528 288no 25 CEP290-323 CEP290-11  990 826 yes 26 CEP290-323 CEP290-252 1173643 no 27 CEP290-323 CEP290-64  1176 640 yes 28 CEP290-323 CEP290-2301609 207 yes 29 Cas9 only wt amplicon = 1816 no 30 GFP only wt amplicon= 1816 no 31 no DNA PCR neg ctrl

Example 5: Gene Expression Analysis of CEP290

Targeted deletion of a region containing the IVS26 splice mutation ispredicted to correct the splicing defect and restore expression of thenormal wild-type CEP290 allele. To quantify expression of the wild-typeand mutant (containing additional cryptic splice mutation) alleles,TaqMan assays were designed. Multiple assays were tested for each RNAspecies and a single wt and single mutant assay were selected. The assayfor the wild-type allele contains a forward primer that anneals in exon26, a reverse primer that anneals in exon 27 and a TaqMan probe thatspans the exon26-exon-27 junction. The assay for the mutant allelecontains a forward primer that anneals in exon 26, a reverse primer thatanneals in the cryptic exon and a TaqMan probe that spans theexon26-cryptic exon junction. A TaqMan assay designed to beta-actin wasused as a control. Total RNA was isolated from IVS26 cells transfectedwith pairs of gRNAs and Cas9-expressing plasmid by either Trizol RNApurification (Ambion), Agencourt RNAdvance (Beckman Coulter) or directcells-to-Ct lysis (Life Technologies). Reverse transcription to generatecDNA was performed and cDNA was used as a template for qRT-PCR usingselected taqman assays on a BioRad real time PCR machine. Relative geneexpression was calculated by ΔΔCt, relative to beta-actin control andGFP-only sample. Increases in expression of wt allele and decreases inexpression of mutant allele relative to GFP-only control indicatecorrected splicing due to gene targeting. FIGS. 12A-12B show initialqRT-PCR data for pairs of gRNAs that had shown activity as eitherindividual gRNAs (measured as described in Example 3) or as pairs(measured as described in Example 4). Pairs of gRNAs that showed thedesired gene expression changes were repeated in replicate experimentsand the cumulative qRT-PCR data is shown in FIG. 13 (error barsrepresent standard error of the mean calculated from 2 to 6 biologicalreplicates per sample).

Example 6: Quantification of Genomic Deletions by ddPCR

Droplet digital PCR (ddPCR) is a method for performing digital PCR inwhich a single PCR reaction is fractionated into 20,000 droplets in awater-oil emulsion and PCR amplification occurs separately in individualdroplets. PCR conditions are optimized for a concentration of DNAtemplate such that each droplet contains either one or no templatemolecules. Assays were designed to perform amplification using BioRadEvaGreen Supermix PCR system with all amplicons ranging in size from250-350 bp. Control assays were designed to amplify segments of theCEP290 gene at least 5 kb away from the IVS26 c.2991+1655A to Gmutation. Assays to detect targeted genomic deletion were designed suchthat amplification of an allele that has undergone deletion will yield aPCR product in the size range of 250-350 bp and amplification will notoccur on a wild-type allele due to the increased distance betweenforward and reverse primers. PCR conditions were optimized on genomicDNA isolated from 293 cells that had been transfected with pairs ofgRNAs and Cas9-expressing plasmid. Deletion assays were verified togenerate no positive signal on genomic DNA isolated from unmodifiedIVS26 fibroblasts. Assays were further tested and optimized on genomicDNA isolated from IVS26 fibroblasts that had been transfected with pairsof gRNAs and Cas9-encoding plasmid. Of the three assays tested for eachof two deletions (CEP290-323 and CEP290-11; and CEP290-323 andCEP290-64) and the 4 control assays tested, a single assay was selectedfor each deletion and a control based on quality data and replicabilityin the ddPCR assay. FIG. 14 shows deletion rates on three biologicalreplicates calculated by taking the number of positive droplets for thedeletion assay and dividing by the number of positive droplets for thecontrol assay.

Example 7: Cloning AAV Expression Vectors

Cloning saCas9 into an AAV Expression Vector

The pAF003 plasmid encodes the CMV-driven S. aureus Cas9 (saCas9), withN-terminal and C-terminal nuclear localization signals (NLS) and aC-terminal triple flag tag, followed by a bovine growth hormone poly(A)tail (bGH polyA). BGH polyA tail was substituted with a 60-bp minimalpolyA tail to obtain pAF003-minimal-pA. The CMV-drivenNLS-saCas9-NLS-3×Flag with the minimal polyA tail was amplified with PCRand subcloned into pTR-UF 11 plasmid (ATCC #MBA-331) with KpnI and SphIsites to obtain the pSS3 (pTR-CMV-saCas9-minimal-pA) vector. The CMVpromoter sequence can be substituted with EFS promoter (pSS10 vector),or tissue-specific promoters (Table 20, e.g. photo-receptor-specificpromoters, e.g. Human GRK1, CRX, NRL, RCVRN promoters, etc.) using SpeIand NotI sites.

Constructing the all-in-One AAV Expression Vector with One gRNA Sequence

For each individual gRNA sequence, a STITCHR product with a U6 promoter,gRNA, and the gRNA scaffold was obtained by PCR with an oligonucleotideencoding the gRNA sequence. The STITCHR product with one dsDNA moleculeof U6-driven gRNA and scaffold was subcloned into pSS3 or pSS10 vectorsusing KpnI sites flanking the STITCHR product and downstream of the leftInverted Terminal Repeat (ITR) in the AAV vectors. The orientation ofthe U6-gRNA-scaffold insertion into pSS3 or pSS10 was determined bySanger sequencing. Alternate promoters may be used to drive gRNAexpression (e.g. H1 promoter, 7SK promoter). Any gRNA scaffold sequencescompatible with Cas variants from other bacterial species could beincorporated into STITCHR products and the AAV expression vectortherein.

Cloning Two gRNA into an AAV Expression Vector

For each pair of gRNA sequences, two ssDNA oligonucleotides weredesigned and obtained as the STITCHR primers, i.e. the left STITCHRprimer and the right STITCHR primer. Two STITCHR PCR reactions (i.e. theleft STITCHR PCR and the right STITCHR PCR) amplified the U6 promoterand the gRNA scaffold with the corresponding STITCHR primer separately.The pSS3 or pSS10 backbone was linearized with KpnI restriction digest.Two dsDNA STITCHR products were purified and subcloned into pSS3 orpSS10 backbone with Gibson Assembly. Due to the unique overlappingsequences upstream and downstream of the STITCHR products, the assemblyis unidirectional. The sequences of the constructs were confirmed bySanger Sequencing. Table 24 lists the names and compositions of AAVexpression vectors constructed, including the names of gRNAs targetinghuman CEP290, the promoter to drive Cas9 expression, and the length ofthe AAV vector including the Inverted Terminal Repeats (ITRs) from wildtype AAV2 genome. Alternative promoters (e.g., H1 promoter or 7SKpromoter) or gRNA scaffold sequences compatible with any Cas variantscould be adapted into this cloning strategy to obtain the correspondingAll-in-One AAV expression vectors with two gRNA sequences.

TABLE 24 Components of AAV expression vectors Promoter Length Name LeftgRNA Right gRNA of saCas9 including ITRs pSS10 NA NA EFS 4100 pSS11CEP290-64  CEP290-323 EFS 4853 pSS15 CEP290-64  NA EFS 4491 pSS17CEP290-323 NA EFS 4491 pSS30 CEP290-323 CEP290-64  EFS 4862 pSS31CEP290-323 CEP290-11  EFS 4862 pSS32 CEP290-490 CEP290-502 EFS 4858pSS33 CEP290-490 CEP290-496 EFS 4858 pSS34 CEP290-490 CEP290-504 EFS4857 pSS35 CEP290-492 CEP290-502 EFS 4858 pSS36 CEP290-492 CEP290-504EFS 4857 pSS3 NA NA CMV 4454 pSS8 CEP290-64  CEP290-323 CMV 5207 pSS47CEP290-323 CEP290-64  CMV 5216 pSS48 CEP290-323 CEP290-11  CMV 5216pSS49 CEP290-490 CEP290-502 CMV 5212 pSS50 CEP290-490 CEP290-496 CMV5212 pSS51 CEP290-490 CEP290-504 CMV 5211 pSS52 CEP290-492 CEP290-502CMV 5212 pSS53 CEP290-492 CEP290-504 CMV 5211 pSS23 NA NA hGRK1 4140pSS24 NA NA hCRX 3961 pSS25 NA NA hNRL 4129 pSS26 NA NA hRCVRN 4083

Example 8: Assessment of the Functions of all-in-One AAV ExpressionVectors

Each individual AAV expression vectors were transfected into 293T cellswith TransIT-293 (Mirus, Inc.) to test their function before beingpackaged into AAV viral vectors. 293T cells were transfected with thesame amount of plasmid and harvested at the same time points. SaCas9protein expression was assessed by western blotting with primaryantibody probing for the triple Flag tag at the C-terminus of saCas9,while loading control was demonstrated by αTubulin expression. Deletionevents at IVS26 mutation could be determined by PCR amplificationfollowed by Sanger sequencing or ddPCR. The results are shown in FIG.15.

Example 9: Production, Purification and Titering of Recombinant AAV2Vectors

Prior to packaging into AAV viral vectors, all AAV expression vector(plasmids) underwent primer walk with Sanger sequencing and functionanalysis. In recombinant AAV (rAAV), two ITRs flanking the transgenecassettes are the only cis-acting elements from the wild-type AAV. Theyare critical for packaging intact rAAVs and genome-release for rAAVvectors during transduction. All AAV expression vectors were restrictiondigested with SmaI or XmaI to ensure the presence of two intact ITRs.

rAAV2 vectors were produced with “Triple Transfection Protocol”: (1) pSSvectors with ITRs and transgene cassettes; (2) pHelper plasmid with E2A,E4, VA genes from Adenovirus; (3) pAAV-RC2 plasmid with Rep and Capgenes from AAV2. These three plasmids were mixed at a mass ratio of3:6:5 and transfected into HEK293 with polymer or lipid-basedtransfection reagent (e.g. PEI, PEI max, Lipofectamine, TransIT-293,etc.). 60-72 hours post-transfection, HEK293 cells were harvested andsonicated to release viral vectors. Cell lysates underwent CsClultracentrifuge to purify and concentrate the viral vectors. Additionalpurification procedures were performed to obtain higher purity forbiophysical assays, including another round of CsCl ultracentrifuge, orsucrose gradient ultracentrifuge, or affinity chromatography. Viralvectors were dialyzed with 1×DPBS twice before being aliquoted forstorage in −80° C. Viral preps can be tittered with Dot-Blot protocolor/and quantitative PCR with probes annealing to sequences on thetransgenes. PCR primer sequences are: AACATGCTACGCAGAGAGGGAGTGG (SEQ IDNO: 399) (ITR-Titer-fwd) and CATGAGACAAGGAACCCCTAGTGATGGAG (SEQ ID NO:400) (ITR-Titer-rev). Reference AAV preps were obtained from the VectorCore at University of North Carolina-Chapel Hill as standards. Toconfirm the presence of three non-structural viral proteins composingthe AAV capsid, viral preps were denatured and probed with anti-AAVVP1/VP2/VP3 monoclonal antibody B1 (American Research Products, Inc. Cat#03-65158) on western blots. The results are shown in FIG. 16.

Example 10: rAAV-Mediated CEP290 Modification In Vitro

293T were transduced with rAAV2 vectors expressing saCas9 with orwithout gRNA sequences to demonstrate the deletion events near the IVS26splicing mutant. 293T cells were transduced with rAAV2 viral vectors atan MOI of 1,000 viral genome (vg)/cell or 10,000 vg/cell and harvestedat three to seven days post transduction. Western blotting with theprimary antibody for Flag (anti-Flag, M2, Sigma-Aldrich) showed that thepresence of U6-gRNA-scaffold does not interfere with saCas9 expression.Genomic DNA from 293T was isolated with the Agencourt DNAdvance Kit(Beckman Coulter). Regions including the deletions were PCR amplifiedfrom genomic DNA isolated, and analyzed on the QIAxcel capillaryelectrophoresis machine. Amplicons smaller than the full-lengthpredicted PCR products represent the deletion events in 293T cells. ThePCR results are shown in FIG. 17. To further understand the nature ofthese deletion events, PCR products were cloned into Zero-Blunt TOPOCloning Kit (Life Technologies) and transformed into chemicallycompetent Top 10 cells. Bacterial colonies were then cultured andsequenced using Sanger sequencing. Sequence results were aligned withthe wt CEP290 locus for analysis.

Example 11: AAV Transduction of Genome Editing Systems in Mouse RetinalExplants

To assess the ability of the AAV vectors described above to transduceCRISPR/Cas9 genome editing systems into retinal cells in situ, an exvivo explant system was developed. FIG. 27A shows a representative imageof an explanted mouse retina on a support matrix, with the tissueindicated by the gray arrow. Explants were harvested at 7- or 10-daytime points, and histological, DNA, RNA and/or protein samples wereproduced. FIG. 27B shows a representative fluorescence micrograph from aretinal explant treated with an AAV vector carrying a GFP reporter,demonstrating successful transduction of an AAV payload in cells inmultiple layers of the retina.

mRNA samples taken from retinal explants further demonstrate that genomeediting systems according to the present disclosure are effectivelytransduced by these AAV vectors: FIG. 28A and FIG. 28B show expressionof Cas9 mRNA and gRNA, respectively, normalized to the expression ofGAPDH. As expected, untreated samples did not express Cas9 or gRNA, andgRNA was not detected in samples that were not transduced with gRNAcoding sequences. Cas9 expression was observed in three AAV constructsin which Cas9 expression was driven by hGRK1, CMV or EFS promoters. Theobservation of Cas9 mRNA and gRNA in samples transduced with vectors inwhich Cas9 expression is driven by the retinal photoreceptor cellspecific hGRK1 promoter indicates that these vectors can transducegenome editing systems in photoreceptor cells in situ.

DNA samples from retinal explants treated with AAV vectors weresequenced, and indel species were identified. The AAV vectors used inthe mouse explant system included guides with targeting domains specificto the mouse CEP290 gene but targeted to the same region of intron 26 asthe human guides presented above; aside from the specific guidesequences used, the AAV vectors used were the same as those describedabove. Table 30 shows a wild type (WT) mouse sequence, with left andright guide sequences italicized, and three representative indels of +1,−4 and −246 aligned with the WT sequence. In the table, three periods (. . . ) represent an abbreviation of the sequence read for ease ofpresentation, while dashes (-) represent alignment gaps and underlinednucleotides represent insertions. Insofar as DNA sequencing of explantstreated with AAV vectors utilizing the photoreceptor specific hGRK1promoter revealed indel formation, these data demonstrate genome editingof a CEP290 target site in retinal photoreceptors.

TABLE 30 Representative Indels in Mouse Retinal Explants WTCCCTCAAACACATGTCTCACGCAGCTTAGACATTCT...CAGAACTCGGTCAG-CATGCTACAGATAGCTTATCT(SEQ ID NO: 2788) (SEQ ID NO: 2789) +1CCCTCAAACACATGTCTCACGCAGCTTAGACATTCT...CAGAACTCGGTCAG GCATGCTACAGATAGCTTATCT (SEQ ID NO: 2788) (SEQ ID NO: 2790) −4CCCTCAAACACATGTCTCACGCAGCTTAGACATTCT...CAGAACTCGG-----CATGCTACAGATAGCTTATCT(SEQ ID NO: 2788) (SEQ ID NO: 2791) −246   CCCTCAAAG---------------------------...---------------CATGCTACAGATAGCTTATCT (SEQID NO: 2792) (SEQ ID NO: 2793)

FIG. 29 summarizes the estimated frequencies of particular editingevents in individual mouse explants transduced with AAV vectorsaccording to the present disclosure. In samples transduced with AAVvectors in which Cas9 expression was driven by the hGRK1 promoter,deletions of sequences between gRNAs (guide sites) were consistentlyobserved, as were indels at one of the two guide sites. Indels at one ofthe two guide sites were also observed in explants transduced with CMVand EFS vectors.

Taken together, these results demonstrate the transduction ofCRISPR/Cas9 genome editing systems into cells, including photoreceptorcells, in the intact mouse retina and the editing (including deletion)of a CEP290 target site in retinal photoreceptors in situ.

Example 12: AAV Transduction of Genome Editing Systems in Primate RetinaIn Vivo

To assess the ability of the AAV vectors described above to transduceCRISPR/Cas9 genome editing systems into retinal cells in vivo, a primatesubretinal injection procedure was developed. Cynomolgus macaquesreceived a bilateral subretinal injections of an AAV5 vector encoding S.aureus Cas9 operably linked to an EFS, CMV or hGRK promoter sequence,and gRNAs C1 and C2, targeted to an intronic region of the cynomolgusCEP290 gene and comprising targeting sequences as set forth in SEQ IDNOs: 2794 and 2796 respectively (see Table 31). AAV injections weregiven at dosages of 4×10¹⁰ (low) or 4×10¹¹ (high) viral genomes (vg).Experimental conditions are summarized in Table 32.

TABLE 31 Cynomolgus gRNA Targeting Domain Sequences Targeting DomainTargeting Domain Guide Sequence (DNA) Sequence (RNA) C1GGCCGGCTAATTTAGTAGAGA GGCCGGCUAAUUUAGUAGAGA (SEQ ID NO: 2794) (SEQ IDNO: 2795) C2 GTTATGAAGAATAATACAAA GUUAUGAAGAAUAAUACAAA (SEQ ID NO: 2796)(SEQ ID NO: 2797)

TABLE 32 Cynomolgus Treatment Conditions Group Vector Dose (vg/eye)CMV-low CEPgRNAs-dCMV-Cas9 4 × 10¹⁰ CMV- CEPgRNAs-dCMV-Cas9 4 × 10¹¹high EFS-low CEPgRNAs-EFS-Cas9 4 × 10¹⁰ EFS-high CEPgRNAs-EFS-Cas9 4 ×10¹¹ GRK-low CEPgRNAs-GRK1-Cas9 4 × 10¹⁰ GRK-high CEPgRNAs-GRK1-Cas9 4 ×10¹¹ Vehicle GRK1-GFP/Vehicle 4 × 10¹¹

6 or 8 mm retinal tissue punches were obtained from AAV-treated andVehicle-treated retinas at 6 and 13 weeks post injection, and genomicDNA was harvested. Sequencing was performed by using a proprietarymethodology (Uni-Directional Targeted Sequencing, or UDiTaS) describedin commonly assigned, copending U.S. Provisional Patent Application No.62/443,212, which is incorporated by reference herein in its entirety.Data from two UDiTaS sequencing reactions with individual upstream ordownstream primers was combined by assuming complete overlap of indelsat the two different gRNA cut sites and by averaging the rates ofinversions and deletions observed in the two sequencing reactions.

Histological analysis demonstrated successful transduction of primatephotoreceptor cells using genome editing systems as disclosed herein.FIG. 27C depicts Cas9 antibody staining in a vehicle-control tissuepunch from a primate retina, while FIG. 27D shows Cas9 expression in apunch from a primate retina treated with an AAV5 vector encoding S.aureus Cas9 operably linked to an hGRK promoter sequence. The figuresshow that the outer nuclear layer (ONL) in the AAV5 vector-treated punchcontains Cas9 protein, while the ONL from the vehicle control punch doesnot. This demonstrates successful transduction of cells in this layer.No detectable Cas9 expression was detected in cells outside the ONL.Because the hGRK promoter is photoreceptor specific, these data indicatethat the systems and methods of this disclosure result in Cas9expression among retinal photoreceptor cells in primates.

FIG. 30 shows the frequency with which specific edits (indels,insertions, deletions and inversions, were observed in each condition.In both the CMV-high and GRK-high conditions, the frequency of editingevents approached or exceeded 40% of reads at the 13-week timepoints.Frequencies of specific edits observed in each experimental condition ateach timepoint are listed in Table 33, below. 13 weeks timepoints forthe EFS-high condition were not obtained.

TABLE 33 Editing Frequencies Observed in Cynomolgus Treatment Conditionsat 6 and 13 Weeks Total Inver- editing sions Deletions Insertions IndelsEFS-low  6 week  2.4% 0.6% 0.4%  0.3%  1.1% 13 week  3.8% 1.2% 0.6% 0.0%  2.0% EFS-high  6 week 10.2% 1.4% 1.3%  2.3%  5.3% 13 week — — — —— CMV-low  6 week  1.1% 0.5% 0.0%  0.1%  0.5% 13 week 13.4% 3.7% 2.1% 0.9%  6.6% CMV-high  6 week  8.0% 0.7% 0.7%  2.1%  4.4% 13 week 44.5%5.1% 3.7% 11.2% 24.5% GRK-low  6 week  5.0% 0.9% 0.7%  0.7%  2.7% 13week  1.6% 0.0% 0.0%  0.3%  1.3% GRK-high  6 week 16.6% 2.5% 2.5%  3.5% 8.1% 13 week 38.0% 7.0% 8.5%  5.9% 16.7%

It should be noted that the hGRK1 promoter is photoreceptor specific,and that the genome editing system encoded by the AAV5 vector would onlybe functional in photoreceptor cells. It is reasonable to conclude,therefore, that the percentages of reads obtained from tissue punches,which include other retinal cell types, are lower than the percentagesthat would be observed in photoreceptor cells alone. Together, thesedata demonstrate transduction of a CRISPR/Cas9 system into a primateretina by subretinal injection of AAV, in vivo, and the generation oftargeted alterations in a CEP290 gene sequence in primate photoreceptorcells in vivo.

Example 13: Correction of IVS26 Splicing Defect by Inversions andDeletions

To verify that deletions and inversions of the intronic region includingthe IVS26 mutation correct the splicing defect observed in CEP290associated disease, a reporter assay was developed utilizing fourreporter constructs having the general design depicted in FIG. 31A:pAD26_SplitGFP+WildType_CEP290_Kan (SEQ ID NO: 2798);pAD27_SplitGFP+Mutant_CEP290_Kan (SEQ ID NO: 2799);pAD28_SplitGFP+Mutant_CEP290_Inverted_Kan (SEQ ID NO: 2800); andpAD29_SplitGFP+DeletionCEP290_Kan (SEQ ID NO: 2801). These constructswere transfected into U20S cells at the concentrations shown in FIG.31B, and GFP and mCherry expression was quantitated for each conditionacross three bioreplicates. Each of the four reporter constructsincluded a sequence encoding a split-green-fluorescent protein (GFP)reporter gene incorporating a 2217 bp human CEP 290 intron sequencecorresponding to (a) wild type (WT), (b) the IVS26 mutation, (c) adeletion of the intronic sequence between two human CEP290 target sites,including the IVS26 mutation and the cryptic exon observed in mRNAs fromsubjects with CEP290 associated disease, as would result from the use ofa genome editing system according to the present disclosure, or (d) aninversion of the intronic sequence between the two human CEP290 targetsites, including the IVS26 mutation and the cryptic exon as would resultfrom the use of a genome editing system of this disclosure. Theconstruct is designed such that correct splicing is necessary for GFPexpression. Thus, the presence of the cryptic splice acceptor site inthe IVS26 condition, but not the WT condition, will result in disruptedGFP transcripts encoding non-functional GFP proteins; modifications atCEP290 target sites that result in the removal or alteration of theIVS26 mutation would rescue the expression of functional GFP protein. Asshown in FIG. 31B, functional GFP protein is expressed at a highbaseline level in cells treated with the WT construct, expression isreduced in the IVS26 condition, and is returned to the WT baseline levelin the deletion and inversion conditions. These data indicate that theaberrant mRNA splicing caused by the IVS26 mutation is rescued by eitherdeletion or inversion of the intronic sequence comprising that mutation.

Example 14: AAV5 Transduction of Genome Editing Systems in Human RetinalExplants

To further establish that the genome editing systems of the presentdisclosure supported targeted gene editing in human retinal cells, e.g.,fully mature human photoreceptors in situ, an ex vivo human retinaexplant system was developed. Purified AAV5 vectors were selected thatencoded S. aureus Cas9 operably linked to an hGRK1 or CMV promotersequence and first and second gRNAs comprising targeting sequencesaccording to SEQ ID NOs: 389 and 388, respectively, and backbonesequences according to SEQ ID NO: 2787. As discussed above, these guidesare targeted to the intronic region of the CEP290 gene on opposite sidesof the IVS26 A>G mutation (Table 28). Human cadaver donor eyes wereobtained within approximately 5 hours post-mortem and 3 mm punches wereimmobilized on a culture substrate as described above. Retinal explantswere treated with AAV vectors at either a low dose of 1×10¹¹ vg or ahigh dose of 5×10¹¹ vg. Experimental conditions are summarized in Table34.

TABLE 34 Human Treatment Conditions Group Vector Dose (vg/punch) CMV-lowCEPgRNAs-dCMV-Cas9 1 × 10¹¹ CMV- CEPgRNAs-dCMV-Cas9 5 × 10¹¹ highGRK-low CEPgRNAs-GRK1-Cas9 1 × 10¹¹ GRK-high CEPgRNAs-GRK1-Cas9 5 × 10¹¹Vehicle GRK1-GFP/Vehicle 5 × 10¹¹

DNA samples from human retinal explants treated with AAV vectors weresequenced at either 14 or 28 days post-transduction, and inversions anddeletions were identified. FIG. 32 summarizes the productive editingobserved in human retinal explants 14 and 28 days after transductionwith the various AAV vectors. Productive editing was defined as totaledits (equal to the sum of the rates of inversions and deletions)capable of correcting the LCA10-associated splice mutation in the CEP290gene (FIG. 32). The most productive editing was observed at 16.4% at the28 day time point for the GRK-high condition. These data demonstratetransduction of a CRISPR/Cas9 system into a human retina by subretinalinjection of AAV and the generation of targeted alterations in a CEP290gene sequence in human photoreceptor cells in situ.

Example 15: AAV5 Transduction of Genome Editing Systems in LiveTransgenic IVS26 Knock-in Mice

To further establish that the genome editing systems of the presentdisclosure supported targeted gene editing of the human CEP290 targetposition in mature photoreceptors in vivo, an IVS26 12 KI mouse modelwas employed. In this model, the human CEP290 exon 26, intron 26 withthe IVS26 mutation (13 c.2991+1655A>G) and exon 27 have been insertedinto the murine CEP290 gene via homologous recombination. AAV5 vectorsencoding (i) S. aureus Cas9 operably linked to thephotoreceptor-specific hGRK1 promoter sequence, and (ii) first andsecond gRNAs comprising targeting sequences according to SEQ ID NOs: 389and 388, respectively, and having gRNA backbone sequences according toSEQ ID NO: 2787 were used as described in Example 14. The vectors wereadministered subretinally (toward the temporal side of the retina nearthe optic nerve) in both eyes of each animal at doses of 1×10¹¹ vg/mL,1×10¹² vg/mL or 1×10¹³ vg/mL; a vehicle group (containing BSS with0.014% Tween20) was also used in the study as a control. Subretinalinjections were conducted in anesthetized mice in accordance with NIHanimal care guidelines. For each injection, a blunt-ended needle(33-gauge, 0.5 in; Hamilton company) on a 5 ml Hamilton syringe wasinserted through the scleral incision, posterior to the lens, and wasadvanced centrally toward the temporal retina until resistance was felt.Care was taken to avoid the damaging the lens as the cannula wasadvanced. A volume of 1 microliter of AAV formulation or vehicle controlcontaining 0.2 mg/mL of fluorescein was injected into the subretinalspace, forming a bleb; fluorescein was used to visualize the bleb and toconfirm successful injection. Animals were euthanized at 6- and 12-weektimepoints, and retinal genomic DNA and RNA were isolated fordetermining the gene editing efficiency (by UDiTaS) and Cas9/gRNA levels(by RT PCR), respectively.

Experimental conditions are summarized in Table 35, along with rates ofinsertion and deletion from individual retinas as measured by UDiTaS.

TABLE 35 Inversion and Deletion Rates in IVS26 KI Mouse Retinas Dose 1 ×10¹² vg 1 × 10¹³ vg Timepoint 6 weeks 12 weeks 6 weeks 12 weeksInversions 4.68% 3.91% 2.06% 1.88% Deletions 6.29% 5.27% 7.79% 4.13%

These data provide further demonstrate the successful transduction ofretinal photoreceptor cells and alteration of the LCA10 target positionusing the vectors and genome editing systems of the present disclosure.

Example 16: Treatment of Inherited Retinal Dystrophy (e.g., LCA) inHumans Using Recombinant Viral Vectors in Combination withCorticosteroids

The recombinant viral vectors disclosed herein may be used for thetreatment of an Inherited Retinal Dystrophy, such as LCA, in a patientin need thereof. In certain embodiments, the patient may have a mutationin the CEP290 gene. For example, the patient may have a mutation inintron 26 (IVS26) of the CEP290 gene (“LCA10-IVS26”). In certainembodiments, the recombinant viral vectors disclosed herein may be usedfor the treatment of Inherited Retinal Dystrophy with CentrosomalProtein 290 (CEP290)-Related Retinal Degeneration Caused by a CompoundHeterozygous or Homozygous Mutation Involving c.2991+1655A>G in Intron26 (IVS26) of the CEP290 Gene in patient in need thereof. Therecombinant viral vector used to treat the patient may be any of the AAVvectors disclosed herein. In certain embodiments, treatment with the AAVvector results in deletion or inversion of the mutation-containingregion in the CEP290 gene, leading to improved photoreceptor and visualfunction. In certain embodiments, the patient may be an adult orpediatric patient.

In certain embodiments, the AAV vector may comprise one or more of theAAV vector genomes as set forth in 19A-F, 20A-20F, 21A-21F, 22A-22F,23A-23F, 24A-24F. In certain embodiments, the AAV vector may compriseone or more of the AAV vector genomes having the configurations asillustrated in FIGS. 25A-25D. In certain embodiments, the AAV vector maycomprise one or more sequences set forth in SEQ ID NOs: 428, 445, 429,446, 430, 447, 431, 448, 432, 449, 433, 450, 2802, or 2803. The AAVvector may comprise a transgene packaged into AAV serotyped with variousAAV capsids, for example, AAV5 capsids. The AAV vector may be formulatedin balanced salt solution/0.001% poloxamer 188 (BSSP).

In certain embodiments, the AAV vector may be administered to thepatient via sub-retinal injection in the para-foveal region. In certainembodiments, the injection may be a single injection. After completionof a pars plana vitrectomy in the eye to be treated, a subretinal blebmay be formed using the AAV vector as the infusion solution.

In certain embodiments, the patient may be administered an AAV vector ata dose of 9×10¹⁰ viral genomes (vg) to 9×10¹¹ vg (e.g., 9×10¹⁰ vg,3×10¹¹ vg, or 9×10¹¹ vg)). For example, in certain embodiments, the AAVvector may be administered to the subject at a dose of 3×10¹¹ vg/ml to3×10¹² vg/ml (e.g., 3×10¹¹ vg/ml, 1×10¹² vg/ml, or 3×10¹² vg/ml). Incertain embodiments, the dose may be administered to the patient in asuitable volume (e.g., 0.3 mL).

To mitigate ocular inflammation and associated discomfort, the patientmay be administered one or more corticosteroids before, at the same timeand/or after administration of the AAV vector. In certain embodiments,the corticosteroid may be an oral corticosteroid (e.g., oralprednisone). In certain embodiments, the patient may be administered thecorticosteroid prior to administration of the AAV vector. For example,the patient may be administered the corticosteroid in the days prior toadministration of the AAV vector (e.g., 3 days prior to administrationof the AAV vector). For example, the corticosteroid treatment may beadministered beginning 3 days prior to until 6 weeks afteradministration of the AAV vector. In certain embodiments, the treatmentof corticosteroids may be 0.5 mg/kg/day for 4 weeks, followed by a15-day taper (0.4 mg/kg/day for 5 days, and then 0.2 mg/kg/day for 5days, and then 0.1 mg/kg/day for 5 days). The dose of corticosteroid maybe changed according to how the patient reacts to the AAV vector. Forexample, if there is an increase in vitreous inflammation by 1+ on thegrading scale while the patient is receiving the 0.5 mg/kg/day dose(i.e., within 4 weeks after surgery), the corticosteroid dose may be maybe increased to 1 mg/kg/day. If any inflammation is present within 4weeks after surgery, the taper may be delayed.

Primary endpoints may include (a) the incidence of dose-limitingtoxicity (DLT), such as macular holes, intraocular inflammation, cornealabrasion, corneal edema, decreased visual acuity, vitreous hemorrhage,subretinal hemorrhage, elevated intraocular pressure, eye pain, eyeirritation, dry eye, retinal detachment, cataract, endophthalmitis, andocular infection; (b) number of adverse events related to the AAVvector; and (c) the number of procedural adverse events. Secondaryendpoints may include one or more of the following: (a) maximumtolerated dose as determined by DLT, (b) change from baseline in (i)mobility course score, (ii) log MAR measurement of best-corrected visualacuity, (iii) the rate of change, decrease in latency, or absolutechange in pupil size following response to light, (iv) dark adaptedvisual sensitivity, (v) the thickness of the outer nuclear layer, (vi)the Pelli-Robson assessment (vii) macular sensitivity, (viii) Farnsworth15 score, (ix) retinal function that is measurable by mfERG, (x)age-range specific quality of life instrument and participant GlobalImpressions of Change and Severity.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

REFERENCES

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What is claimed is:
 1. A method of treating LCA10 in a subject in needthereof comprising administering to the subject: an adeno-associated(AAV) vector comprising a nucleotide sequence encoding a first guide RNA(gRNA) molecule comprising a first targeting domain complementary with afirst target domain from the CEP290 gene and a nucleotide sequenceencoding a Cas9 molecule; wherein the AAV vector is administered to theeye of the subject, wherein the AAV vector is capable of delivery to anon-dividing cell, and wherein the administration results inNHEJ-mediated alteration of an LCA10 target position in one or morecells of the subject thereby treating the subject; and a corticosteroidadministered daily at a first dose for at least 4 weeks beginning nomore than fourteen days prior to administering the AAV vector.
 2. Themethod of claim 1, wherein the AAV vector further comprises a nucleotidesequence encoding a second gRNA molecule comprising a second targetingdomain complementary with a second target domain from the CEP290 gene.3. The method of claim 2, wherein the corticosteroid is administered atthe first dose beginning one, two, three, four, five, or six days priorto administering the AAV vector.
 4. The method of claim 3, wherein thecorticosteroid is an oral corticosteroid.
 5. The method of claim 4,wherein the oral corticosteroid is prednisone.
 6. The method of claim 5,wherein the first dose of the corticosteroid is 0.1 mg/kg/per day to 1mg/kg/day.
 7. The method of claim 6, wherein the first targeting domaincomprises a nucleotide sequence selected from the group consisting ofSEQ ID NO:530 (CEP290-323), SEQ ID NO:555 (CEP290-485), SEQ ID NO:468(CEP290-490), and SEQ ID NO:538 (CEP290-492).
 8. The method of claim 7,wherein the second targeting domain comprises a nucleotide sequenceselected from the group consisting of SEQ ID NO:558 (CEP290-64), SEQ IDNO:2321 (CEP290-11), SEQ ID NO:2312 (CEP290-230), SEQ ID NO:460(CEP290-496), SEQ ID NO:586 (CEP290-502), and SEQ ID NO:568(CEP290-504).
 9. A method of treating LCA10 in a subject in need thereofcomprising administering to the subject: a recombinant viral particlecomprising a nucleotide sequence encoding a first guide RNA (gRNA)molecule comprising a first targeting domain complementary with a firsttarget domain from the CEP290 gene, a nucleotide sequence encoding asecond gRNA molecule comprising a second targeting domain complementarywith a second target domain from the CEP290 gene; and a nucleotidesequence encoding a Cas9 molecule; wherein the recombinant viralparticle is administered to the eye of the subject, and wherein therecombinant viral particle is capable of delivery to a non-dividingcell, and wherein the administration results in NHEJ-mediated alterationof an LCA10 target position in one or more cells of the subject therebytreating the subject; and a corticosteroid administered daily at a firstdose for at least 4 weeks beginning no more than fourteen days prior toadministering the recombinant viral particle.
 10. The method of claim 9,wherein the corticosteroid is administered at the first dose beginningone, two, three, four, five, or six days prior to administering therecombinant viral particle.
 11. The method of claim 10, wherein thefirst dose of the corticosteroid is 0.1 mg/kg/per day to 1 mg/kg/day.12. The method of claim 11, wherein the corticosteroid is an oralcorticosteroid.
 13. The method of claim 12, wherein the oralcorticosteroid is prednisone.
 14. The method of claim 13, wherein thefirst targeting domain comprises a nucleotide sequence selected from thegroup consisting of SEQ ID NO:530 (CEP290-323), SEQ ID NO:555(CEP290-485), SEQ ID NO:468 (CEP290-490), and SEQ ID NO:538(CEP290-492).
 15. The method of claim 14, wherein the second targetingdomain comprises a nucleotide sequence selected from the groupconsisting of SEQ ID NO:558 (CEP290-64), SEQ ID NO:2321 (CEP290-11), SEQID NO:2312 (CEP290-230), SEQ ID NO:460 (CEP290-496), SEQ ID NO:586(CEP290-502), and SEQ ID NO:568 (CEP290-504).
 16. A method of treatingLCA10 in a subject in need thereof comprising administering to thesubject: an AAV vector comprising a nucleotide sequence encoding a firstguide RNA (gRNA) molecule, a nucleotide sequence encoding a second gRNAmolecule and a nucleotide sequence encoding a Cas9 molecule, wherein theAAV vector is administered to the eye of the subject, wherein the firstand the second gRNA molecules are configured to form a first and asecond complex with the Cas9 molecule, respectively, and wherein thefirst complex forms a first DNA double strand break between the 5′ endof the Alu repeat at c.2991+1162 to c.2991.1638 of intron 26 of theCEP290 gene and 500 nucleotides upstream of said 5′ end of the Alurepeat, and the second complex forms a second DNA double strand breakbetween an LCA10 target position and 500 nucleotides downstream of theLCA10 target position, wherein the first and second DNA double strandbreaks are repaired by NHEJ in a manner that results in alteration ofthe LCA10 target position thereby treating the subject; and acorticosteroid administered daily at a first dose for at least 4 weeksbeginning no more than fourteen days prior to administering the AAVvector.
 17. The method of claim 16, wherein the corticosteroid is anoral corticosteroid.
 18. The method of claim 17, wherein the oralcorticosteroid is prednisone.
 19. The method of claim 18, wherein thecorticosteroid is administered at the first dose beginning one, two,three, four, five, or six days prior to administering the AAV vector.20. The method of claim 19, wherein the AAV vector is an AAV5 vector.21. The method of claim 20, wherein the corticosteroid is administeredat a dose of 0.1 mg/kg/per day to 1 mg/kg/day.
 22. The method of claim21, wherein the nucleotide sequence encoding a first gRNA moleculecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NO:530 (CEP290-323), SEQ ID NO:555 (CEP290-485), SEQ ID NO:468(CEP290-490), and SEQ ID NO:538 (CEP290-492).
 23. The method of claim22, wherein the nucleotide sequence encoding a second gRNA moleculecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NO:558 (CEP290-64), SEQ ID NO:2321 (CEP290-11), SEQ ID NO:2312(CEP290-230), SEQ ID NO:460 (CEP290-496), SEQ ID NO:586 (CEP290-502),and SEQ ID NO:568 (CEP290-504).
 24. The method of claim 8, wherein theAAV vector is administered to the subretinal space of the eye.
 25. Themethod of claim 24, wherein (i) the corticosteroid is administered dailyat a first dose for at least four weeks; (ii) the corticosteroid isadministered daily at a second dose for five days after administrationof the first dose, wherein the concentration of the second dose is lowerthan the concentration of the first dose; (iii) the corticosteroid isadministered daily at a third dose for five days after administration ofthe second dose, wherein the concentration of the third dose is lowerthan the concentration of the second dose; and (iv) the corticosteroidis administered daily at a fourth dose for five days afteradministration of the third dose, wherein the concentration of thefourth dose is lower than the concentration of the third dose.
 26. Themethod of claim 25, wherein (i) the first dose is 0.5 mg/kg; (ii) thesecond dose is 0.4 mg/kg; (iii) the third dose is 0.2 mg/kg; and (iii)the fourth dose is 0.1 mg/kg.
 27. The method of claim 24, wherein thecorticosteroid is administered at a first dose of 1.0 mg/kg.
 28. Themethod of claim 15, wherein the recombinant viral particle isadministered to the subretinal space of the eye.
 29. The method of claim28, wherein (i) the corticosteroid is administered daily at a first dosefor at least four weeks; (ii) the corticosteroid is administered dailyat a second dose for five days after administration of the first dose,wherein the concentration of the second dose is lower than theconcentration of the first dose; (iii) the corticosteroid isadministered daily at a third dose for five days after administration ofthe second dose, wherein the concentration of the third dose is lowerthan the concentration of the second dose; and (iv) the corticosteroidis administered daily at a fourth dose for five days afteradministration of the third dose, wherein the concentration of thefourth dose is lower than the concentration of the third dose.
 30. Themethod of claim 29, wherein (i) the first dose is 0.5 mg/kg; (ii) thesecond dose is 0.4 mg/kg; (iii) the third dose is 0.2 mg/kg; and (iii)the fourth dose is 0.1 mg/kg.
 31. The method of claim 28, wherein thecorticosteroid is administered at a first dose of 1.0 mg/kg.
 32. Themethod of claim 23, wherein the AAV vector is administered to thesubretinal space of the eye.
 33. The method of claim 32, wherein (i) thecorticosteroid is administered daily at a first dose for at least fourweeks; (ii) the corticosteroid is administered daily at a second dosefor five days after administration of the first dose, wherein theconcentration of the second dose is lower than the concentration of thefirst dose; (iii) the corticosteroid is administered daily at a thirddose for five days after administration of the second dose, wherein theconcentration of the third dose is lower than the concentration of thesecond dose; and (iv) the corticosteroid is administered daily at afourth dose for five days after administration of the third dose,wherein the concentration of the fourth dose is lower than theconcentration of the third dose.
 34. The method of claim 33, wherein (i)the first dose is 0.5 mg/kg; (ii) the second dose is 0.4 mg/kg; (iii)the third dose is 0.2 mg/kg; and (iii) the fourth dose is 0.1 mg/kg. 35.The method of claim 32, wherein the corticosteroid is administered at afirst dose of 1.0 mg/kg.